This invention relates to a novel protein having a lecithin-cholesterol acyltransferase-like activity, etc. or its salt, a precursor protein of the protein or its salt, a partial peptide of the protein or its salt; a DNA coding for the protein; a recombinant vector; a transformant; a method for producing the protein, a pharmaceutical composition comprising the protein, the partial peptide or its salt; and an antibody to the protein or the partial peptide. The protein, the partial peptide or its salt, and the DNA are useful as an agent for treating or preventing arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity or hypertriglyceridemia. The antibody can be used in assay of the protein, the partial peptide or its salt. The protein, the partial peptide or its salt is useful as a reagent for the screening for candidate medical compounds.

The present invention relates to a novel protein having a lecithin-cholesterol acyltransferase-like activity etc. and a DNA coding for the protein.
Cholesterol is an important lipid constituting the animal cell membrane and defining its character. Moreover, it is a precursor of steroid hormones, thus being a substance essential to animal life. However, due to the recent changes in dietary habit and ecology, arteriosclerosis and other adult diseases arising from pathological intracellular accumulation of cholesterol are now presenting a serious problem so that elucidation of the mechanisms of cholesterol metabolism in the body is being awaited.
In the efflux of cholesterol from the peripheral cells, high density lipoprotein (hereinafter sometimes referred to briefly as HDL) is suspected to play a cardinal role and this assumption has been supported by the epidemiologic finding of an inverse correlation between risk for coronary artery disease and plasma HDL levels and the experimental finding that HDL in culture medium stimulates cholesterol efflux from cells and decreases the intracellular concentration of cholesterol (Journal of Lipid Research, 37, 2473, 1996). In the reverse cholesterol transport system, lecithin-cholesterol acyltransferase (hereinafter sometimes referred to briefly as LCAT) is involved to a significant extent.
LCAT transfers the xcex2-acyl group (fatty acid) of lecithin (phosphatidylcholine) to the 3xcex2-OH group of cholesterol, so that it consumes the equivalent moles of lecithin and unesterified cholesterol and produces the equivalent moles of cholesteryl ester and lysolecithin (Journal of Lipid Research, 9, 155, 1968). In the circulation, most of LCAT exists in HDL to show activity and a portion of the cholesteryl ester produced in the HDL is taken up and metabolized in the liver, while another portion of the ester migrates into the nonpolar core of the HDL particle to give rise to a mature HDL rich in cholesteryl esters. Owing to the concentration gradient resulting from the consumption of unesterified cholesterol in HDL, the HDL continuously absorbs cholesterol from the other cell membranes. In this manner, LCAT together with HDL is in charge of the reverse cholesterol transport from peripheral tissues to the liver, thus contributing to the anti-atherosclerotic action of HDL (Biochimica et Biophysica Acta, 1084, 205, 1991).
In familial LCAT deficiency which is an inheritable disease, the reverse cholesterol transport system is lacking so that characteristic tissue damages occur from deposits of cholesterol, leading to coroneal opacity, hemolytic anemia associated with a morphological abnormality of erythrocytes, and proteinuria and renal failure due to kidney impairment (Lancet, 388, 778, 1991). In addition to gene abnormalities, various illnesses involving plasma lipid abnormalities cause changes in LCAT activity. For example, LCAT activity is reportedly elevated by hypercalorism or in obesity and hypertriglyceridemia (Clinical Science, 38, 593, 1970) and decreased in malnutrition, abetalipoproteinemia, and Tangier disease.
LCAT is a 416-residue polypeptide synthesized in the liver, and exists as a glycoprotein with a molecular mass of 59-68 KDa (Journal of Biological Chemistry, 254, 7456, 1979). In the blood, most of LCAT exists in HDL and in the expression of its activity, Apo AI, the principal apoprotein of HDL, acts as the cofactor to stimulate LCAT activity (FEBS Letters, 15, 355, 1971). There exist a variety of mutant LCAT genes corresponding to the variation in enzyme defect and clinical picture in various cases of familial LCAT activity deficiency, and they are discharging significant functions in the metabolism of plasma lipoproteins.
So far, only one kind of LCAT has been reported and the existence of any analogous protein having similar activity has not been predicted.
Meanwhile, in arteriosclerosis, thrombus formation, and post-PTCA restenosis, abnormality of vascular tonus, enhancement of inflammatory reactions, and abnormality of the coagulation-fibrinolysis system, which stem from endothelial cell impairment, occur to cause a remodeling of the blood vessel with the proliferation and transformation of vascular smooth muscle cells as a cardinal pathological picture. The changes on the molecular level which occur in the course of formation of vascular lesions are now being understood in terms of a group of transcription factors controlling the expression of individual genes (Kurabayashi et al., Modern Medicine, 52, 2340, 1997). In such a specifically expressed gene, there is a promoter (enhancer-repressor) sequence which functions only ad hoc and this promoter domain controls the transcription levels of the mRNA encoding the protein.
Some of those promoters are known to be hormone-dependent or growth factor-dependent, and by utilizing them, several drug screening systems and transgenic animals have already been created and actually the systems used in the screening for drugs and the animals used in the analysis of vital functions.
The present invention has for its object to provide a novel protein having LCAT and other activities, a precursor protein thereof, a partial peptide, a salt of either of them, a signal peptide, a DNA coding for said protein, precursor protein, partial peptide or signal peptide, a recombinant vector, a transformant, a method of producing said protein, a pharmaceutical composition comprising said protein or DNA, an antibody against said protein, a screening method/screening kit for a compound promoting or inhibiting the LCAT activity of said protein, a compound obtained by using said screening method, a pharmaceutical composition comprising said compound, a promoter for a novel protein having LCAT and other activities, a screening method/screening kit for a compound promoting or inhibiting the promoter activity, a compound obtained by using the screening method mentioned just above, and a pharmaceutical composition comprising the compound.
The inventors of the present invention did intensive research to accomplish the above-mentioned objects and succeeded in cloning a cDNA having a novel nucleotide sequence from each of human heart-, human kidney-, and mouse kidney-derived cDNA libraries, and discovered that the protein encoded by those cDNA clones is a lecithin-cholesterol acyltransferase-like protein (hereinafter sometimes referred to as the LCAT-like protein). The inventors further cloned the genomic DNA of said LCAT-like protein and by a promoter activity assay, found a promoter for said LCAT-like protein.
The present invention, provides:
(1) a protein comprising an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or a substantial equivalent thereto, a precursor protein thereof or a salt thereof,
(2) a protein or precursor protein according to the above item (1), which comprises an amino acid sequence represented by any one of SEQ ID NO:4 to SEQ ID NO:8,
(3) a protein or precursor protein according to the above item (1), which possesses lecithin-cholesterol acyltransferase-like activity,
(4) a partial peptide of the protein according to the above item (1), or a salt thereof,
(5) a signal peptide comprising an amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, or a substantial equivalent thereto,
(6) a DNA which comprises a DNA having a nucleotide sequence coding for the protein or precursor protein according to the above item (1),
(7) a DNA according to the above item (6), which comprises a nucleotide sequence represented by any one of SEQ ID NO:12 to SEQ ID NO:19,
(8) a DNA which comprises a DNA having a nucleotide sequence coding for the signal peptide according to the above item (5),
(9) a DNA according to the above item (8), which comprises a nucleotide sequence represented by SEQ ID NO:20 to SEQ ID NO:22,
(10) a recombinant vector comprising the DNA according to the above item (6),
(11) a transformant which is transformed by the recombinant vector according to the above item (10),
(12) a method for producing the protein or the precursor protein according to the above item (1) or the salt thereof, which comprises cultivating the transformant according to the above item (11) to produce and accumulate the protein or the precursor protein according to the above item (1), and collecting the same,
(13) a pharmaceutical composition which comprises the protein or the precursor protein or a salt thereof according to the above item (1),
(14) a pharmaceutical composition which comprises the DNA according to the above item (6),
(15) a pharmaceutical composition according to the above item (13) or the above item (14), which is an agent for treating or preventing arteriosclerosis, atherosclerosis hyperlipidemia, obesity, inflammatory diseases, senescence, diseases of brain or renal disorder,
(16) an antibody against (i) the protein or the precursor protein according to the above item (1), (ii) the partial peptide according to the above item (4), or (iii) a salt thereof,
(17) a method for screening for a compound which promotes or inhibits a lecithin-cholesterol acyltransferase-like activity of (i) the protein according to the above item (1), (ii) the partial peptide according to the above item (4), or (iii) a salt thereof, which comprises using (i) the protein according to the above item (1), (ii) the partial peptide according to the above item (4), or (iii) a salt thereof,
(18) a kit for screening for a compound which promotes or inhibits a lecithin-cholesterol acyltransferase-like activity of(i) the protein according to the above item (1), (ii) the partial peptide according to the above item (4), or (iii) a salt thereof, which comprises using (i) the protein according to the above item (1), (ii) the partial peptide according to the above item (4), or (iii) a salt thereof,
(19) a compound which promotes or inhibits a lecithin-cholesterol acyltransferase-like activity of (i) the protein according to the above item (1), (ii) the partial peptide according to the above item (4), or (iii) a salt thereof, which is obtained by using the screening method according to the above item (17) or the screening kit according to the above item (18),
(20) a pharmaceutical composition which comprises a compound which promotes or inhibits a lecithin-cholesterol acyltransferase-like activity of (i) the protein according to the above item (1), (ii) the partial peptide according to the above item (4), or (iii) a salt thereof, which is obtained by using the screening method according to the above item (17) or the screening kit according to the above item (18),
(21) a DNA which comprises a promoter DNA having a nucleotide sequence represented by SEQ ID NO:38 or a substantial equivalent thereto or its partial DNA having a promoter activity, and
(22) a method for screening for a compound or a salt thereof which promotes or inhibits the promoter activity of the DNA according to the above item (21). The present invention, furthermore, provides:
(23) a protein or the precursor according to the above item (1), wherein the amino acid sequence is an amino acid sequence having an identity of not less than about 50% (preferably not less than about 60%, more preferably not less than about 70%, still more preferably not less than about 80%, still more preferably not less than about 90%, most preferably not less than about 95%) to the total amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or a substantial equivalent thereto,
(24) a protein or the precursor according to the above item (1), which comprises (i) an amino acid sequence wherein one or more amino acid residues (for example about 1 to 30 amino acid residues) are deleted from the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a substantial equivalent thereto, (ii) an amino acid sequence wherein one or more amino acid residues (for example about 1 to 30 amino acid residues) are added to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a substantial equivalent thereto, (iii) an amino acid sequence wherein one or more amino acid residues (for example 1 to 30 amino acid residues). are inserted into the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a substantial equivalent thereto, (iv) an amino acid sequence wherein one or more other amino acid residues in the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or a substantial equivalent thereto, are substituted with 1 or more amino acid residues (for example about 1 to 30), or (vi) combinations thereof,
(25) a peptide according to the above item (4), which comprises at least one amino acid sequence selected from the group consisting of amino acid sequences of the 3rd-25th residues, 27th-36th residues, 43rd-66th residues, 68th-86th residues, 92nd-98th residues,107th-153rd residues, 155th-168th residues, 172nd-180th residues, 189th-240th residues, 256th-262nd residues, 268th-275th residues, 277th-287th residues, 295th-306th residues 308th-332nd residues, 336th-347th residues and/or 351st-377th residues of the amino acid sequence represented by SEQ ID NO:1,
(26) an isolated DNA comprising a DNA having a nucleotide sequence which hybridizes under highstringent condition to a nucleotide sequence represented by any one of SEQ ID NO:12 to SEQ ID NO:19,
(27) a recombinant vector comprising the DNA according to the above item (26),
(28) a transformant which is transformed with the recombinant vector according to the above item (27),
(29) a process for producing a protein, a precursor thereto, or a salt thereof which comprises culturing the transformant according to the above item (28) under conditions suitable to express and accumulate the protein, a precursor thereto, or a salt thereof which is encoded by the DNA according to the above item (26) or a salt thereof and collect the same,
(30) a protein, a precursor thereto encoded by the DNA according to the above item (26) which is produced by the process according to the above item (29) or a salt thereof,
(31) a method according to the above item (17), which comprises measuring and comparing a lecithin-cholesterol acyltransferase-like activity, in cases that (i) the protein according to the above item (1), the partial peptide according to the above item (4), or a salt thereof is contacted with a lecithin and a non-esterified cholesterol, and (ii) the protein according to the above item (1), the partial peptide according to the above item (4),or a salt thereof is contacted with a lecithin, a non-esterified cholesterol and a test compound,
(32) a pharmaceutical composition which comprises a compound or a salt thereof having a promoting activity of a lecithin-cholesterol acyltransferase-like activity of the protein according to the above item (1), the partial peptide according to the above item (4) or a salt thereof, obtained by the screening method according to the above item (17) or the screening kit according to the above item (18),
(33) a pharmaceutical composition according to the above item (32), which is an agent for treating or preventing arteriosclerosis, atherosclerosis, hyperlipidemia, obesity, atherosclerosis senescence, diseases of brain or renal disorder,
(34) a pharmaceutical composition according to the above item (32), which is an agent for treating or preventing hypercalorism, obesity or hypertriglyceridemia,
(35) a pharmaceutical composition which comprises a compound or a salt thereof having a inhibiting activity of a lecithin-cholesterol acyltransferase-like activity of the protein according to the above item (1), the partial peptide according to the above item (4) or a salt thereof, obtained by the screening method according to the above item (17), or the screening kit according to the above item (18),
(36) a pharmaceutical composition according to the above item (35), which is an agent for treating or preventing malnutrition, abetalipoproteinemia, inflammatory diseases or Tangier disease,
(37) a method of quantitative determination of the protein according to the above item (1), the partial peptide according to the above item (4) or a salt thereof in a test liquid sample, which comprises (a) competitively reacting the test liquid sample and a labeled protein according to the above item (1) partial peptide according to the above item (4) or a salt thereof with the antibody according to the above item (16), and (b) measuring the ratio of the labeled protein according to the above item (1), partial peptide according to the above item (4) or a salt thereof which binds to the antibody,
(38) a method of quantitative determination of the protein according to the above item (1), the partial peptide according to the above item (4) or a salt thereof in a test liquid sample, which comprises (a) reacting the test liquid sample with the antibody according to the above item (16) immobilized on an insoluble carrier and another antibody which is labeled according to the above item (16) simultaneously or continuously, and (b) measuring the activity of the labeling agent on the insoluble carrier,
(39) a pharmaceutical composition which comprises the antibody according to the above item (16),
(40) a pharmaceutical composition according to the above item (39), which is an agent for treating or preventing malnutrition, abetalipoproteinemia inflammatory diseases or Tangier disease,
(41) an antisense DNA having a nucleotide sequence complementary or substantially complementary to the DNA according to the above item (4) or the above item (26), and capable of suppressing expression of the same DNA,
(42) an antisense DNA according to the above item (41), wherein the nucleotide sequence substantially complementary to the DNA according to the above item (4) or (26) is a nucleotide sequence having an identity of not less than about 70% (preferably not less than about 90%, more preferably not less than about 95%) to the total nucleotide sequence or partial nucleotide sequence of the nucleotide sequence complementary to the DNA according to the above item (4) or (26),
(43) a pharmaceutical composition which comprises the antisense DNA according to the above item (41), and
(44) an pharmaceutical composition according to the above item (43), which is an agent for treating or preventing malnutrition, abetalipoproteinemia inflammatory diseases or Tangier disease.
The protein (the protein of the invention) having an amino acid sequence identical or substantially equivalent thereto identical to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 may be any of the proteins derived from various tissues of man and other warm-blooded animals (e.g. guinea pig, rat, mouse, fowl, rabbit, swine, sheep, bovine, monkey, etc.). Among such tissues are cells (e.g. hepatocytes, splenocytes, nerve cells, glia cells, xcex2 cells of pancreas, myelocytes, mesangial cells, Langerhan"" cells, epidermic cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, myocites, adipocytes, immune cells (e.g. macrophages, T-cells, B cells, natural killer cells, mastocytes, neutrophils, basophils, eosinophils, monocytes), megarocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes, interstitial cells, the corresponding precursor cells, stem cells, cancer cells, etc.), all tissues in which such cells exist, for example the brain, various parts of the brain (e.g. olefactory bulb, amygdaloid body, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medula oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow, adrenal, skin, muscle, lung, bowels (e.g. large intestine and small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., and blood series cells and the corresponding cultured cell lines (e.g. MEL, Ml, CTLL-2, HT-2, WEHI-3, HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG-01, etc.). The protein mentioned above may also be a synthetic protein.
The amino acid sequence represented by SEQ ID NO:2 is an amino acid sequence in which 32 amino acid residues of the 64th (Glu) to 95th (Leu) amino acid residues of SEQ ID NO:2 is inserted between the 63rd (Leu) and 64th (Val) amino acids of SEQ ID NO:1.
Examples of the amino acid sequence which is substantially equivalent to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 are an amino acid sequence which is not less than about 50%, preferably not less than about 60%, more preferably not less than about 70%, still more preferably not less than about 80%, still more preferably not less than about 90%, and most preferably not less than about 95% identity to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and so on.
And, as an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, an amino acid sequence containing the amino acid sequence of the 3rd-25th residues, 27th-36th residues, 43rd-66th residues, 68th-86th residues, 92nd-98th residues, 107th-153rd residues, 155th-168th residues, 172nd-180th residues, 189th-240th residues, 256th-262nd residues, 268th-275th residues, 277th-287th residues, 295th-306th residues, 308th-332nd residues, 336th-347th residues, and/or 351st-377th residues, respectively, of the amino acid sequence represented by SEQ ID NO:1, and so on are also preferred.
Examples of the protein of the present invention which comprises an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 are a protein having an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and having a qualitatively equivalent activity to the protein having the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
Examples of the lecithin:cholesterol acyltransferase-like activity are lecithin:cholesterol acyltransferase activity, phospholipase activity, lysophosphatidylcholine-to-phosphatidylcholine esterification activity, phospholipid:cholesterol acyttransferase activity, lysophospholipase activity, PAF hydrolysis/transesterification activity, fatty acid ester hydrolyzing activity, phospholipid oxide hydrolyzing and cholesterol oxide esterification activity, high-density lipoprotein (HDL) metabolism regulation activity, and lipase activity.
The term xe2x80x9cqualitatively equivalent activityxe2x80x9d is used herein to mean substantial equivalence in qualitative terms such as a lecithin-cholesterol acyltransferase (LCAT)-like activity, etc. Therefore, the degree of equivalence may range, for example, from about 0.1 to about 100 times (preferably about 0.5 to 10, more preferably 0.5 to 2 times). However, differences in quantitative terms such as the potency of activity and the molecular mass of protein are immaterial.
Activities such as a LCAT-like activity may be measured by a per se known method or its analogue method. For example, the activities may be measured by the method for screening as mentioned below.
And, the protein of the present invention includes the so called muteins, for example, proteins comprising (1) an amino acid sequence wherein one or more amino acid residues (preferably 1 to 30, more preferably about 1 to 10, and still more preferably a few (1 to 5) amino acid residues) are deleted from the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, (2) an amino acid sequence wherein one or more amino acid residues (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably a few (1 to 5) amino acid residues) are added to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, (3) an amino acid sequence wherein one or more amino acid residues (preferably 1 to 30, more preferably about 1 to 10, and still more preferably a few (1 to 5) amino acid residues) are inserted into the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, (4) an amino acid sequence wherein one or more other amino acid residues in the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 are substituted with 1 or more amino acid residues (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably a few (1 to 5) amino acid residues), or (5) combinations thereof.
In the above-mentioned deletion, substitution, or insertion, examples of the positions of deletion, substitution or insertion are not so critical but are preferably positions other than the 3rd-25th residues, 27th-36th residues, 43rd-66th residues, 68th-86th residues, 92nd-98th residues, 107th-153rd residues, 155th-168th residues, 172nd-180th residues, 189th-240th residues, 256th-262nd residues, 268th-275th residues, 277th-287th residues, 295th-306th residues, 308th-332nd residues, 336th-347th residues or 351st-377th residues of the amino acid sequence represented by SEQ ID NO:1, and are not so critical but are preferably positions other than the corresponding position of SEQ ID NO:2.
In the above-mentioned deletion, substitution, or insertion, another example of the positions of deletion, substitution, or insertion are not so critical but are preferably positions other than (1) the 163rd(Ala)-167th(Gly) residues of the amino acid sequence represented by SEQ ID NO:1, (2) the 195th(Ala)-199th(Gly) residues of the amino acid sequence represented by SEQ ID NO:2, (3) the 96th(Val)-127th(Asp) residues of the amino acid sequence represented by SEQ ID NO:2, (4) the 163rd(Ala)-167th(Gly) residues of the amino acid sequence represented by SEQ ID NO:3, etc. The 163rd(Ala)-167th(Gly) residues of the amino acid sequence represented by SEQ ID NO:1 is a central active amino acid residues of the protein of the present invention.
Moreover, in the above-mentioned deletion or substitution, example of the positions of deletion or substitution are not so critical but are preferably positions of the 2nd, 26th, 37th, 42nd, 67th, 87th, 91st, 99th-100th, 106th, 154th, 169th, 171st, 181st, 188th, 241st, 245th, 251st, 255th, 263rd-264th, 267th, 276th, 288th-289th, 294th, 307th, 333rd, 335th, 348th, 350th or 378th of the amino acid sequence represented by SEQ ID NO:1 or SEQ ID NO:3, or the corresponding position of SEQ ID NO:2.
On the other hand, in the above-mentioned addition, example of the positions of addition are not so critical but are (1) the amino acid sequence represented by SEQ ID NO:4, wherein three amino acids are added to the N-terminal amino acid sequence represented by SEQ ID NO:1, or (2) the amino acid sequence represented by SEQ ID NO: 5, wherein three amino acids are added to the N-terminal amino acid sequence represented by SEQ ID NO:2, etc.
Accordingly, the protein of the present invention may include a protein having an amino acid sequence represented by SEQ ID NO:4 or SEQ ID NO:5, or substantially equivalent thereof. In this case, the term xe2x80x9csubstantially equivalentxe2x80x9d means the same as mentioned above, The amino acid sequence represented by SEQ ID NO:5 is an amino acid sequence in which 32 amino acid residues of the 67th(Glu)-98th(Leu) in SEQ ID NO:5 is inserted between the 66th(Leu) and the 67th(Val) in SEQ ID NO:4.
Throughout this specification, proteins are represented in accordance with the conventions for description of peptides, that is the N-terminus (amino terminus) at left and the C-terminus (carboxyl terminus) at right. The protein of the present invention including the protein containing the amino acid sequence of SEQ ID NO:1 is usually in the carboxyl (xe2x80x94COOH) or carboxylate (xe2x80x94COO ) form at the C-terminus but may be in the amide (xe2x80x94CONH2) or ester (xe2x80x94COOR) form.
R in the ester residue includes a C1-6 alkyl group (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.), a C3-8 cycloalkyl group (e.g. cyclopentyl, cyclohexyl, etc.), a C6-12 aryl group (e.g. phenyl, xcex1-naphthyl, etc.), a C7-14 aralkyl group such as a phenyl-C1-2 alkyl group (e.g. benzyl, phenethyl, etc.) and xcex1-naphthyl-C1-2 alkyl, (e.g. xcex1-naphthylmethyl, etc.), as well as pivaloyloxymethyl group which is often used for the production of esters for oral administration.
When the protein of the present invention has a carboxyl or a carboxylate group in any position other than the C-terminus, the corresponding carboxamide or ester form is also included in the scope of the present invention. The ester mentioned just above such an esters mentioned for the C-terminal carboxyl group.
Furthermore, the protein of the present invention includes (1) the protein in which the N-terminus amino acid residue is protected with a protective group (e.g. C1-6 acyl group such as C1-6 alkanoyl such as formyl, acetyl, etc.), (2) the protein in which the N-terminal side of Gln has been cleaved in vivo to form pyroglutamic acid, (3) the protein in which a side chain of any relevant constituent amino acid (e.g. xe2x80x94OH, xe2x80x94SH, xe2x80x94NH2, imidazole group, indole group, guanidino group, etc.) is protected by any protective group (e.g. C1-6 acyl group such as C1-6 alkanoyl such as formyl or acetyl, etc.), and (4) a complex protein such as glycoproteins obtained by attachment of sugar chains.
Examples of the protein of the present invention are (1) a protein which is derived from human-heart and comprises the amino acid sequence represented by SEQ ID NO:1 or SEQ ID NO:4 (FIG. 1), (2) a protein which is derived from human-kidney and comprises the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:5 (FIG. 2), and a protein which is derived from murine-kidney and comprises the amino acid sequence represented by SEQ ID NO:3 (FIG. 3).
The precursor protein of the present invention, for example, is a protein in which one or more amino acids, preferably 1 to 10 amino acids, more preferably 1 to 100 amino acids, and still more preferably 1 to 200 amino acids are added to the N-terminal and/or C-terminal amino acid of the above-mentioned protein of the present invention.
Examples of the precursor protein of the present invention are a protein which comprises the amino acid sequence represented by SEQ ID NO:6, the amino acid sequence represented by SEQ ID NO:7 or the amino acid sequence represented by SEQ ID NO:8, or a substantial equivalent thereto.
Further, the precursor protein of the present invention may be any of the proteins derived from various tissues of man and other warm-blooded animals (e.g. guinea pig, rat, mouse, fowl, rabbit, swine, sheep, bovine, monkey, etc.), and also the precursor protein of the present invention may be a synthetic protein.
Examples of the precursor protein of the present invention are a protein which comprises an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 and comprises an amino acid sequence which is not less than about 50%, preferably not less than about 60%, more preferably not less than about 70%, still more preferably not less than about 80%, still more preferably not less than about 90%, and most preferably not less than about 95% identity to the amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and a protein which is able to produce the protein of the present invention. Therefore, the quantitative equivalency, such as a molecular weight, is not always necessary.
The precursor protein of the present invention may include any peptides comprising (1) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) are deleted from the amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, (2) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) are added to the amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, (3) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) are inserted into the amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, (4) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) are substituted with the amino acid sequence represented by SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (4) combinations thereof.
In the above-mentioned insertion, deletion or substitution are not so critical but are preferably positions other than (1) the 196th (Ala)-200th (Gly) residues of the amino acid sequence represented by SEQ ID NO:6, (2) the 228th (Ala)-232nd (Gly) residues of the amino acid sequence represented by SEQ ID NO:7, (3) the 129th (Val)-160th (Asp) residues of the amino acid sequence represented by SEQ ID NO:7, (4) the 196th (Ala)-200th (Gly) residues of the amino acid sequence represented by SEQ ID NO:8, and so on.
The precursor protein of the present invention including the protein which is usually in the carboxyl (xe2x80x94COOH) or carboxylate (xe2x80x94COOxe2x88x92) form at the C-terminus but may be in the amide (xe2x80x94CONH2) or ester (xe2x80x94COOR) form, as above-mentioned protein of the present invention.
Furthermore, the precursor protein of the present invention includes (1) the protein in which the N-terminus amino acid residue (e.g. methionine residue) is protected with a protective group (e.g. C1-6 acyl group such as C1-6 alkanoyl such as formyl, acetyl, etc.), (2) the protein in which the N-terminal side of Gln has been cleaved in vivo to form pyroglutamic acid, (3) the protein in which the side chain of any relevant constituent amino acid (e.g. xe2x80x94OH, xe2x80x94SH, xe2x80x94NH2, imidazole group, indole group, guanidino group, etc.) has been protected by any protective group (e.g. C1-6 acyl group such as C1-6 alkanoyl such as formyl or acetyl, etc.), and (4) a complex protein such as glycoproteins obtained by attachment of sugar chains, as above-mentioned protein of the present invention.
Examples of the precursor protein of the present invention are (1) the protein having the amino acid sequence represented by SEQ ID NO.:6, that is a protein having the amino acid sequence represented by SEQ ID NO:1 to which the signal peptide having the amino acid sequence-represented by SEQ ID NO:10 is added to N-terminus amino acid, (2) the protein having the amino acid sequence represented by SEQ ID:7, that is a protein having the amino acid sequence represented by SEQ ID NO:2 to which the signal peptide having the amino acid sequence represented by SEQ ID NO:10 is added to N-terminus amino acid, and so on.
The amino acid sequence represented by SEQ ID NO:7 is an amino acid sequence wherein the 32 amino acid residues(i.e. the 97th(Glu)-128th(Leu) residues) of the amino acid sequence represented by SEQ ID NO:7 is inserted between the 96th(Leu) and 97th(Val) of the amino acid sequence represented by SEQ ID NO:7.
As the precursor protein of the present invention has a signal peptide, the precursor protein of the present invention is able to secrete the protein of the present invention as a secreted protein efficiently. Also the precursor protein is useful as an intermediate for producing the protein of the present invention.
Moreover, as the precursor protein of the present invention is able to possess the same function as that of the protein of the present invention, the precursor protein of the present invention has the same usefulness as the protein of the present invention.
The partial peptide of the protein of the present invention may be any peptide having a qualitatively If equivalent activity to the above-mentioned protein of the present invention such as a LCAT-like activity and so on. For example, the partial peptides include peptides comprising at least not less than about 20, preferably not less than about 50, more preferably not less than about 70, for still better result, not less than about 100, best result, not less than 200 amino acid residues of the amino acid sequence of the proteins of the present invention.
Examples of the partial peptide are a peptide comprises amino acid sequences selected from the amino acid sequences corresponding to the 3rd-25th residues, 27th-36th residues, 43rd-66th residues, 68th-86th residues, 92nd-98th residues, 107th-153rd residues, 155th-168th residues, 172nd-180th residues, 189th-240th residues, 256th-262nd residues, 268th-275th residues, 277th-287th residues, 295th-306th residues, 308th-332nd residues, 336th-347th residues and/or 351st-377th residues of the amino acid sequence represented by SEQ ID NO:1.
A preferable example of the partial peptide is a which comprises an amino acid sequence corresponding to the 163rd (Ala)-167th(Gly) residues of the amino acid sequence represented by SEQ ID NO:1 (that is, the 195th(Ala)-199th (Gly) residues of the amino acid sequence represented by SEQ ID NO:2, the 163rd(Ala)-167th(Gly) residues of the amino acid sequence represented by SEQ ID NO:3).
The partial peptide of the present invention includes the so called muteins, for example, proteins comprising (1) an amino acid sequence wherein one or more amino acid residues (preferably about 1 to 10, more preferably about a few (1 to 5) amino acid residues) are deleted from the above-mentioned partial peptide, (2) an amino acid sequence wherein one or more amino acid residues (preferably about 1 to 10, more preferably a few (1 to 5) amino acid residues) are added to the above-mentioned partial peptide, (3) an amino acid sequence wherein one or more amino acid residues (preferably about 1 to 10, more preferably a few (1 to 5) amino acid residues) are inserted into the above-mentioned partial peptide, (4) an amino acid sequence wherein one or more other amino acid residues in the above-mentioned partial peptide are substituted with 1 or more amino acid residues (preferably about 1 to 10, and more preferably a few (1 to 5) amino acid residues), or (5) combinations thereof.
The partial peptide of the present invention including the peptide which is usually in the carboxyl (xe2x80x94COOH) or carboxylate (xe2x80x94COOxe2x88x92) form at the C-terminus but may be in the amide (xe2x80x94CONH2) or ester (xe2x80x94COOR) form, as above-mentioned protein of the present invention.
Furthermore, the partial peptide of the present invention includes (1) the peptide in which the N-terminus amino acid residue (e.g. methionine residue) has been protected with a protective group (e.g. C1-6 acyl group such as C1-6 alkanoyl such as formyl, acetyl, etc.), (2) the peptide in which the N-terminal side of Gln is cleaved in vivo to form pyroglutamic acid, (3) the peptide in which the side chain of any relevant constituent amino acid (e.g. xe2x80x94OH, xe2x80x94SH, xe2x80x94NH2, imidazole group, indole group, guanidino group, etc.) is protected by any protective group (e.g. C1-6 acyl group such as C1-6 alkanoyl such as formyl or acetyl, etc.), and (4) a complex peptide such as glycoproteins obtained by attachment of sugar chains, as above-mentioned protein of the present invention.
Moreover, the partial peptide of the present invention is not always necessary to have a LCAT activity as the partial peptide of the present invention is able to be used as an antigen for producing an antibody.
Examples of the signal peptide of the present invention are a peptide which comprises the amino acid sequence represented by SEQ ID NO:9, the amino acid sequence represented by SEQ ID NO:10 or the amino acid sequence represented by SEQ ID NO:11, or a substantial equivalent thereto.
Further, the signal peptide of the present invention may be any of the proteins derived from various tissues of man and other warm-blooded animals (e.g. guinea pig, rat, mouse, fowl, rabbit, swine, sheep, bovine, monkey, etc.), and also the signal peptide of the present invention may be a synthetic peptide.
Examples of the signal peptide of the present invention are a peptide which comprises an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11 and comprises an amino acid sequence which is not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, still more preferably not less than about 80%, still more preferably not less than about 90%, and most preferably not less than about 95% identity to the amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, and a peptide comprises an amino acid sequence substantially equivalent to the amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, and has a function as an signal peptide. Therefore, the quantitative equivalency, such as a molecular weight, is not always necessary.
The signal peptide of the present invention may include any peptides comprising (1) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 10, preferably 1 to 5, more preferably a few (1 to 3) amino acid residues) are deleted from the amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, (2) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 10, preferably 1 to 5, more preferably a few (1 to 3) amino acid residues) are added to the amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, (3) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 10, A preferably 1 to 5, more preferably a few (1 to 3) amino acid residues) are inserted into the amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, (4) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 10, preferably 1 to 5, more preferably a few (1 to 3) amino acid residues) are substituted with the amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, or (5) combinations thereof.
The signal peptide of the present invention includes the peptide which is usually in the carboxyl (xe2x80x94COOH) or carboxylate (xe2x80x94COOxe2x88x92) form at the C-terminus but may be in the amide (xe2x80x94CONH2) or ester (xe2x80x94COOR) form, as above-mentioned protein of the present invention.
Furthermore, the signal peptide of the present invention includes (1) the peptide in which the N-terminus amino acid residue (e.g. methionine residue) is protected with a protective group (e.g. C1-6 acyl group such as C1-6 alkanoyl such as formyl, acetyl, etc.), (2) the peptide in which the N-terminal side of Gln is cleaved in vivo to form pyroglutamic acid, (3) the peptide in which the side chain of any relevant constituent amino acid (e.g. xe2x80x94OH, xe2x80x94SH, xe2x80x94NH2, imidazole group, indole group, guanidino group, etc.) is protected by any protective group (e.g. C1-6 acyl group such as C1-6 alkanoyl such as formyl or acetyl, etc.), and (4) a complex peptide such as glycoproteins obtained by attachment of sugar chains, as above-mentioned protein of the present invention.
Examples of the signal peptide of the present invention are (1) a peptide comprising the amino acid sequence represented by SEQ ID NO:9, wherein the protein of the present invention comprises the amino acid sequence represented by SEQ ID NO:4 is deleted from the precursor protein of the present invention comprising the amino acid sequence represented by SEQ ID NO:6, (2) a peptide comprising the amino acid sequence represented by SEQ ID NO:10, wherein the protein of the present invention comprising the amino acid sequence represented by SEQ ID NO:1 is deleted from the precursor protein of the present invention comprises the amino acid sequence represented by SEQ ID NO:6, and (3) a peptide comprises the amino acid sequence represented by SEQ ID NO:11, wherein the protein of the present invention comprises the amino acid sequence represented by SEQ ID NO:3 is deleted from the precursor protein of the present invention comprising the amino acid sequence represented by SEQ ID NO:8, and so on.
The signal peptide of the present invention is able to secrete various kinds of secreted proteins, including the protein of the present invention, efficiently.
The salts of the protein, the precursor protein, the partial peptide or the signal peptide of the present invention includes salts with physiologically acceptable bases (e.g. alkali metals) or acids such as organic or inorganic acids, and is preferably a physiologically acceptable acid addition salt. Examples of such salts are salts thereof with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid, etc.) and salts thereof with organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid, etc.)
The protein, the precursor protein or a salt thereof of the present invention can be produced from the tissues or cells of human or other warm-blooded animals by per se known purification techniques or, as described hereinafter, by culturing a transformant carrying a DNA encoding the protein. It can also be produced in accordance with the procedures for peptide synthesis which are described hereinafter.
When the protein or the precursor protein of the present invention is produced from the tissues or cells of human or other warm-blooded animals, the tissues or cells of human or other warm-blood animals are homogenized and the protein of the present invention is extracted by an acid, etc. The protein can be isolated and purified from the extract by a combination of chromatography such as reverse phase chromatography, ion exchange chromatography and so on.
For the synthesis of the protein, the precursor protein, the partial peptide, the signal peptide or their salts, or their amide form of the present invention, any of commercial resins available for protein synthesis can be employed. Among such resins are chloromethyl resin, hydroxymethyl resin, benzhydrylamino resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamino resin, PAM resin, 4-hydroxymethyl-methylphenylacetamidomethyl resin, polyacrylamide resin, 4-(2xe2x80x2,4xe2x80x2-dimethoxyphenyl-hydroxymethyl)phenoxy resin, and 4-(2xe2x80x2,4xe2x80x2-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin. Using such a resin, amino acids which may be beforehand protected at side-chain functional groups in a suitable manner can be serially condensed with the xcex1-amino group in the order corresponding to the amino acid sequence of the objective protein by various condensation techniques which are per se known. After completion of the final condensation reaction, the protein is separated from the resin and the protective groups are removed. Then, in highly diluted solution, the intramolecular disulfide-forming reaction is carried out to provide the objective proteins or amides thereof.
Referring to the above condensation of protected amino acids, various activating agents known to be useful for protein synthesis can be utilized, and carbodiimide reagents are especially preferred. The carbodiimide reagents include DCC, N,Nxe2x80x2-diisopropylcarbodiimide, N-ethyl-Nxe2x80x2-(3-dimethylaminoprolyl)carbodiimide and so on. For activation by these reagents, the protected amino acid and a racemization inhibitor (e.g. HOBt, HOOBt, etc.) can be directly added to the resin, or the protected amino acid can be activated beforehand in the form of symmetric acid anhydride, HOBt ester or HOOBt ester and, then, added to the resin.
The solvent used for the above-mentioned activation of protected amino acids or a conjugation thereof to the resin can be properly selected from among the solvents known to be useful for protein condensation reactions. Examples of the solvent are acid amides (e.g. N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.), halogenated hydrocarbons (e.g. methylene chloride, chloroform, etc.), alcohols (e.g. trifluoroethanol, etc.), sulfoxides (e.g. dimethyl sulfoxide, etc.), ethers (e.g. dioxane, tetrahydrofuran, etc.), nitrites (e.g. acetonitrile, propionitrile, etc.), esters (e.g. methyl acetate, ethyl acetate, etc.), and suitable mixtures of these solvents. The reaction temperature can be selected from the range known to be useful for protein-forming reactions, usually the range of about xe2x88x9220xc2x0 C. to about 50xc2x0 C. The activated amino acid derivative is generally used in a 1.5 to 4-fold excess. When the condensation is found insufficient by ninhydrin assay, the reaction can be repeated to make the condensation thoroughly sufficient. When sufficient condensation can not be achieved by repeated reaction, an unreacted amino acid can be acetylated by using acetic anhydride or acetylimidazole so as not to effect a subsequent reaction.
The protective groups for protecting the amino group of the starting compound include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxy-benzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, and so on.
The carboxyl group can be protected in the form of, for example, an alkyl ester (e.g. straight-chain, branched, or cyclic alkyl esters such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, and so on), an aralkyl ester (e.g. benzyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, benzhydryl, and so on), phenacyl ester, benzyloxycarbonylhydrazide, t-butoxycarbonylhydrazide or tritylhydrazide.
The hydroxyl group of serine can be protected in the form of an ester or an ether. The group suitable for esterification includes carboxylic acid-derived acyl groups such as a lower(C1-6) alkanoyl group (e.g. acetyl, etc.), an aroyl group (e.g. benzoyl, etc.), a benzyloxycarbonyl, an ethoxycarbonyl group and so on. The group suitable for etherification includes a benzyl group, a tetrahydropyranyl group, a t-butyl group and so on.
The protective group used for protecting the phenolic hydroxyl group of tyrosine includes Bzl, C12-Bzl, 2-nitrobenzyl, Br-Z, t-butyl and so on.
The protective group for the imidazole group of histidine includes Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and so on.
The starting compound with activated carboxyl groups includes the corresponding acid anhydride, azide, and active ester (e.g. esters with alcohols such as pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt, etc.). The starting compound with activated amino groups includes the corresponding phosphorylamide.
The method for removal of such protective groups includes catalytic reduction in a hydrogen stream in the presence of a catalyst (e.g. Pd black or Pd-on-carbon), acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, treatment with a base such as diiso-propylethylamine, triethylamine, piperidine, piperazine or the like, and reduction with sodium metal in liquid ammonia. The above deprotection by treatment with acid is generally conducted at a temperature of about xe2x88x9220xc2x0 C. to 40xc2x0 C. This acid treatment can be carried out advantageously in the presence of a cation acceptor such as anisole, phenol, thioanisole, m-cresol, p-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, or the like. The 2,4-dinitrophenyl group used for protecting the imidazole group of histidine can be removed by treatment with thiophenol, and the formyl group used for protecting the indole group of tryptophan can be removed not only by said acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol or the like as described hereinbefore, but also by alkali treatment with diluted sodium hydroxide solution, diluted liquid ammonia, or the like.
The method for protecting any functional group that should not take part in the contemplated reaction, the protective group to be used for such protection, the method for eliminating the protective group, and the method for activating the functional group to be involved in the contemplated reaction can all be properly selected from among the known methods and groups.
An alternative method for providing the protein in amide form typically comprises protecting the xcex1-carboxyl group of the C-terminal amino acid in the form of an amide, extending the peptide (protein) chain to a desired length towards the N-terminus, deprotecting the N-terminal xcex1-amino acid of the resulting peptide chain selectively to provide an N-terminal-deprotected fragment, preparing a peptide (protein) fragment with its C-terminal carboxyl group selectively deprotected, and condensing the two fragments in a solvent such as the mixed solvent as mentioned above. The condensation reaction can be carried out in the same manner as described hereinbefore. After purification of the protected protein thus obtained by condensation, all the protective groups are eliminated by the procedures described hereinbefore to provide the contemplated protein in a crude form. This crude protein is purified by suitable known purification techniques and lyophilized to provide the desired protein amide.
A method for providing the protein in an ester form comprises condensing the xcex1-carboxyl group of the C-terminal amino acid with a suitable alcohol to prepare the corresponding ester and subjecting this ester to the same procedure as described for purification of the protein amide to provide the objective protein ester.
The partial peptide, the signal peptide of the present invention or a salt thereof can be produced by per se known procedures for peptide synthesis or by cleaving the protein or the precursor protein of the present invention with a suitable peptidase. The process for peptide synthesis may be a solid-phase synthesis and/or a liquid-phase synthesis. Namely, the objective peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein with the residual part thereof and, when the product has a protective group, the protective group is removed whereupon a desired peptide can be manufactured. The known technology for condensation and deprotection includes the procedures described in the following literature (1)-(5).
(1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York, 1966
(2) Schroeder and Luebke, The Peptide, Academic Press, New York, 1965
(3) Nobuo Izumiya et al., Fundamentals and Experiments in Peptide Synthesis, Maruzen, 1975
(4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment Series 1, Protein Chemistry IV, 205, 1977
(5) Haruaki Yajima (ed.), Development of Drugs-Continued, 14, Peptide Synthesis, Hirokawa Shoten
After the reaction, the partial peptide of the present invention can be isolated and purified by a combination of conventional purification techniques such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide isolated as above is in a free form, it can be converted to a suitable salt by known methods or method analogous thereto. On the other hand, when it is isolated as a salt, it can be converted to a free form or to another salt by known methods or method analogous thereto.
The DNA coding for the protein of the present invention may be any DNA comprising a nucleotide sequence encoding the protein of the present invention as mentioned above. It may also be any one of genomic DNA, genomic DNA library, cDNA derived from the tissues or cells as mentioned above, cDNA library derived from the tissues or cells as mentioned above, and synthetic DNA.
The vector for constructing a library may include bacteriophage, plasmid, cosmid, and phagemid. Furthermore, using a total RNA fraction or an mRNA fraction prepared from the tissues or cells, a direct amplification can be carried out by Reverse Transcriptase Polymerase Chain Reaction (hereinafter, referred to as RT-PCR method) technique.
Examples of the DNA coding for the protein of the present invention are (1) a DNA comprising a nucleotide sequence represented by SEQ ID NO:12, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:12 under a highstringent condition and codes for a protein having a substantially equivalent activity (e.g. LCAT activity etc.) to the protein of the present invention, (2) a DNA comprising a nucleotide sequence represented by SEQ ID NO:13, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:13 under a highstringent condition and codes for a protein having a substantially equivalent activity (e.g. LCAT activity etc.) to the protein of the present invention, (3) a DNA comprising a nucleotide sequence represented by SEQ ID NO:14, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:14 under a highstringent condition and codes for a protein having a substantially equivalent activity (e.g. LCAT activity etc.) to the protein of the present invention, (4) a DNA comprising a nucleotide sequence represented by SEQ ID NO:15, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:15 under a highstringent condition and codes for a protein having a substantially equivalent activity (e.g. LCAT activity etc.) to the protein of the present invention, or (5) a DNA comprising a nucleotide sequence represented by SEQ ID NO:16, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:16 under a highstringent condition and codes for a protein having a substantially equivalent activity (e.g. LCAT activity etc.) to the protein of the present invention, and so on.
Examples of the DNA which comprises the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:12 to SEQ ID NO:16 under a highstringent condition are a DNA comprising a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:12 to SEQ ID NO:16.
The hybridization can be carried out by per se known methods such as the method described in Molecular Cloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) and so on. When a commercially available library is used, the hybridization can be carried out in accordance with the instructions given in the accompanying manual, and particularly, be carried out under a highstringent condition.
Under the highstringent condition, Na+ concentration is at about 19 to 40 mM, preferably about 19 to 20 mM and a temperature is at about 50 to 70xc2x0 C., preferably about 60 to 65xc2x0 C. Particularly, the condition at about 19 mM of Na+ and about 65xc2x0 C. is preferred.
Preferable examples of the DNA coding for the protein comprising the amino acid sequence represented by SEQ ID NO:1 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:12.
Preferable examples of the DNA comprising the DNA coding for the protein having the amino acid sequence represented by SEQ ID NO:2 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:13, and so on.
Preferable examples of the DNA comprising the DNA coding for the protein having the amino acid sequence represented by SEQ ID NO:3 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:14, and so on.
Preferable examples of the DNA comprising the DNA coding for the protein having the amino acid sequence represented by SEQ ID NO:4 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:15, and so on.
Preferable examples of the DNA comprising the DNA coding for the protein having the amino acid sequence represented by SEQ ID NO:5 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:16, and so on.
The DNA coding for the precursor protein of the present invention may be any DNA comprising a nucleotide sequence encoding the precursor protein of the present invention as mentioned above. It may also be any one of genomic DNA, genomic DNA library, cDNA derived from the tissues or cells as mentioned above, cDNA library derived from the tissues or cells as mentioned above, and synthetic DNA.
Examples of the DNA coding for the precursor protein of the present invention are (1) a DNA comprising a nucleotide sequence represented by SEQ ID NO:17, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:17 under a highstringent condition and codes for a protein which is able to produce the above-mentioned precursor protein of the present invention, (2) a DNA comprising a nucleotide sequence represented by SEQ ID NO:18, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:18 under a highstringent condition and codes for a protein which is able to produce the above-mentioned precursor protein of the present invention, or (3) a DNA comprising a nucleotide sequence represented by SEQ ID NO:19, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:19 under a highstringent condition and codes for a protein which is able to produce the above-mentioned precursor protein of the present invention, and so on.
Examples of the DNA which comprises the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:17 to SEQ ID NO:19 under a highstringent condition are a DNA comprising a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:17 to SEQ ID NO:19.
The method of the hybridization and the highstringent condition are the same as mentioned-above.
A preferable example of the DNA comprising the DNA coding for the precursor protein having the amino acid sequence represented by SEQ ID NO:6 is a DNA comprising the nucleotide sequence represented by SEQ ID NO:17, and so on.
A preferable example of the DNA comprising the DNA coding for the precursor protein having the amino acid sequence represented by SEQ ID NO:7 is a DNA comprising the nucleotide sequence represented by SEQ ID NO:18, and so on.
A preferable example of the DNA comprising the DNA coding for the precursor protein having the amino acid sequence represented by SEQ ID NO:8 is a DNA comprising the nucleotide sequence represented by SEQ ID NO:19, and so on.
The DNA coding for the precursor protein of the present invention may be any DNA comprising a nucleotide sequence encoding the precursor protein of the present invention as mentioned above. It may also be any one of genomic DNA, genomic DNA library, cDNA derived from the tissues or cells as mentioned above, cDNA library derived from the tissues or cells as mentioned above, and synthetic DNA.
Examples of the DNA coding for the partial peptide of the present invention are (1) a DNA comprising a nucleotidze sequence represented by SEQ ID NO:12, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:12 under a highstringent condition and comprising a partial nucleotide sequence of a nucleotide sequence coding for the protein of the present invention or a substantial equivalent thereto, (2) a DNA comprising a nucleotide sequence represented by SEQ ID NO:13, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:13 under a highstringent condition and comprising a partial nucleotide sequence of a nucleotide sequence coding for the protein of the present invention or a substantial equivalent thereto, (3) a DNA comprising a nucleotide sequence represented by SEQ ID NO:14, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:14 under a highstringent condition and comprising a partial nucleotide sequence of a nucleotide sequence coding for the protein of the present invention or a substantial equivalent thereto, (4) a DNA comprising a nucleotide sequence represented by SEQ ID NO:15, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:15 under a highstringent condition and comprising a partial nucleotide sequence of a nucleotide sequence coding for the protein of the present invention or a substantially equivalent thereto, or (5) a DNA comprising a nucleotide sequence represented by SEQ ID NO:15, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:15 under a highstringent condition and comprising a partial nucleotide sequence of a nucleotide sequence coding for the protein of the present invention or a substantially equivalent thereto, and so on.
Examples of the DNA which comprises the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:12 to SEQ ID NO:16 under a highstringent condition are a DNA comprising a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:12 to SEQ ID NO:16.
The method of the hybridization and the highstringent condition are the same as mentioned-above.
Specifically, examples of the DNA coding for the partial peptide having at least one amino acid sequence selected from the amino acid sequences of the 3rd-25th amino acid residues, 27th-36th residues, 43rd-66th residues, 68th-86th residues, 92nd-98th residues, 107th-153rd residues, 155th-168th residues, 172nd-180th residues, 189th-240th residues, 256th-262nd residues, 268th-275th residues, 277th-287th residues and 295th-306th residues, 308th-332nd, 336th-347th and/or 351st-377th respectively, of the amino acid sequence represented by SEQ ID NO:1 are a DNA having at least one nucleotide sequence selected from sequences of the 7th-75th nucleotide sequence, 79th-108th nucleotide sequence, 127th-198th nucleotide sequence, 202nd-258th nucleotide sequence, 274th-294th nucleotide sequence, 319th-459th nucleotide sequence, 463rd-504th nucleotide sequence, 514th-540th nucleotide sequence, 565th-720th nucleotide sequence, 766th-786th nucleotide sequence, 802nd-825th nucleotide sequence, 829th-861st nucleotide sequence, 883rd-918th nucleotide sequence, 922nd-996th nucleotide sequence, 1006th-1041st nucleotide sequence, and/or 1051st-1131st nucleotide sequence, respectively, of the nucleotide sequence represented by SEQ ID NO:12 and so on.
The DNA coding for the signal peptide of the present invention may be any DNA comprising a nucleotide sequence encoding the signal peptide of the present invention as mentioned above. It may also be any one of genomic DNA, genomic DNA library, cDNA derived from the tissues or cells as mentioned above, cDNA library derived from the tissues or cells as mentioned above, and synthetic DNA.
Examples of the DNA coding for the signal peptide of the present invention are (1) a DNA comprising a nucleotide sequence represented by SEQ ID NO:20, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:20 under a highstringent condition and coding for the peptide having a function as a signal peptide, (2) a DNA comprising a nucleotide sequence represented by SEQ ID NO:21, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:21 under a highstringent condition and coding for the peptide having a function as a signal peptide, (3) a DNA comprising a nucleotide sequence represented by SEQ ID NO:22, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:22 under a highstringent condition and coding for the peptide having a function as a signal peptide, and so on.
Examples of the DNA which comprises the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:20 to SEQ ID NO:22 under a highstringent condition are a DNA comprising a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:20 to SEQ ID NO:22.
The method of the hybridization and the highstringent condition are the same as mentioned-above.
A preferable example of the DNA comprising the DNA coding for the signal peptide having the amino acid sequence represented by SEQ ID NO:9 is a DNA comprising the nucleotide sequence represented by SEQ ID NO:20, and so on.
A preferable example of the DNA comprising the DNA coding for the signal peptide having the amino acid sequence represented by SEQ ID NO:10 is a DNA comprising the nucleotide sequence represented by SEQ ID NO:21, and so on.
A preferable example of the DNA comprising the DNA coding for the signal peptide having the amino acid sequence represented by SEQ ID NO:11 is a DNA comprising the nucleotide sequence represented by SEQ ID NO:22, and so on.
The DNA encoding the protein, the precursor protein, the partial peptide or the signal peptide of the present invention (hereinafter, these proteins or peptides are sometimes referred to briefly as the protein of the present invention) can be cloned either by PCR amplification using synthetic DNA primers having a partial nucleotide sequence of the DNA coding for the protein or by hybridization using the DNA inserted in a suitable vector and labeled DNA fragment or synthetic DNA coding for a part or full region of the protein or the partial peptide of the present invention. The hybridization can be carried out by the method described in Molecular Cloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available DNA library is used, the instructions given in the accompanying manual can be followed.
The substitution of the nucleotide sequence of the DNA can be carried out by the per se known method such as Gapped duplex method, Kunkel method and so on by using the known kits such as MutanTM-G (Takara corporation), MutanTM-K (Takara corporation) and so on.
The cloned DNA coding for the protein or the partial peptide of the present invention can be used directly or after digestion with a restriction enzyme or after addition of a linker depending on purposes. This DNA may have ATG as the translation initiation codon at the 5xe2x80x2 end and TAA, TGA, or TAG as the termination codon at the 3xe2x80x2 end. The translation initiation and termination codons can be added by means of suitable DNA adapters.
An expression vector for the protein of the present invention can be constructed by, for example, (a) cutting out an objective DNA fragment from the DNA for the protein of the present invention and (b) ligating the objective DNA fragment with the downstream of a promoter in a suitable expression vector.
The vector may include plasmids derived from Escherichia coli, e.g., pBR322, pBR325, pUC12, pUC13, etc.; plasmids derived from Bacillus subtilis, e.g., puB110, pTP5, pC194, etc.; plasmids derived from yeasts e.g., pSH19, pSH15, etc.; bacteriophages such as xcex-phage: animal virus such as retrovirus, vaccinia virus, etc.; insect virus; and other vectors such as pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo and so on.
According to the present invention, any promoter can be used as long as it is appropriate for the host cell which is used for expressing a gene. When the host is an animal cell, the promoter includes SR xcex1, SV40 promoter, LTR promoter, CMV(cytomegalovirus) promoter, HSV-TK promoter, etc., and CMV promoter and SR xcex1 promoter are preferably used. When the host for the transformation is Escherichia coli, the promoter is preferably trp promoter, lac promoter, recA promoter, xcexPL promoter, lpp promoter, T7 promoter, etc. When the host for the transformation is Bacillus, the promoter is preferably SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is a yeast, the promoter is preferably PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. When the host is an insect cell, the promoter include polyhedrin promoter, P10 promoter, etc.
The expression vectors may, if necessary, further comprise enhancers, splicing signals, polyadenylation signals, selective markers, SV40 duplicate origin (hereinafter referred to as SV40 ori). Examples of selective markers are dihydrofolate reductase (hereinafter referred to as dhfr gene, ampicillin resistant gene (hereinafter referred to as Ampr), neomycin-resistant gene (hereinafter referred to as Neor) and so on. The dhfr gene gives methotrexate (MTX) resistant and Neo gives G418 resistant. Particularly, when the dhfr gene is used as a selective marker against dhfr gene-deficient chinese hamster cell line, cells transfected by the objective gene can be selected in a thymidine-free medium.
Furthermore, an appropriate signal sequence for a host can be added to the N-terminal side of the protein. When the host is Escherichia coli, the utilizable signal sequences may include PhoA signal sequence, OmpA signal sequence, etc. When the host is Bacillus, they may include xcex1-amylase signal sequence, subtilisin signal sequence, etc. When the host is a yeast, they may include MFxcex1 signal sequence, SUC2 signal sequence, etc. When the host is an animal cell, they may include insulin signal sequence, xcex1-interferon signal sequence, antibody molecule signal sequence, etc.
A transformant or transfectant is obtained by using the vector thus constructed, which carries the DNA coding for the protein of the present invention.
The host may be, for example, Escherichia species, Bacillus species, yeast cells, insect cells, insects, animal cells, etc.
Examples of Escherichia species include Escherichia coli K12.DH1 (Proceedings of the National Academy of Sciences of the United State of America, Vol. 60, 160 (1968)), JM103 (Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology, Vol. 120, 517 (1978)), HB101 (Journal of molecular Biology, Vol, 41, 459 (1969)), C600 [Genetics, Vol. 39, 440 (1954)), etc.
Examples of Bacillus species are, for example, Bacillus subtilis MI114 (Gene, Vol. 24, 255 (1983)), 207-21 (Journal of Biochemistry, Vol. 95, 87 (1984)), etc.
Examples of yeast cells are, for example, Saccharomyces cerevisiae AH22, AH22Rxe2x88x92, NA87-11A, DKD-5D or 20B-12, Schizosachcaromyces pombe NCYC1913 or Pichia pastoris KM71, etc.
Examples of insect cells are, for example, Spodoptera frugiperda cell (Sf cell), MG1 cell derived from a center intestine of Trichoplusia ni, High Five(trademark) cell derived from eggs of Trichoplusia ni, Mamestra brassicae-derived cell, Estiamena acrea-derived cell and so on when virus is AcNPV; and Bombvx mori N cell (BmN cell,) and so on when virus is BmNPV. Examples of the Sf cell are, for example, Sf9 cell (ATCC CRL 1711), Sf21 cell [both, Vaughn J. L. et al., In Vivo, 13, 213-217(1977)] and so on.
Examples of insects include a larva of silkworm (Bombyx mori larva) (Maeda et al., Nature, 315, 592(1985)).
Examples of animal cells are, for example, monkey-derived COS-7 cell line, Vero cell line, Chinese hamster ovary cell line (hereinafter referred to as CHO cell), dhfr gene-deficient Chinese hamster cell line (hereinafter referred to as CHO(dhfrxe2x88x92) cell), mouse L cell, mouse AtT-20, mouse myeloma cell, rat GH3, human FL, 293 cell, C127 cell, BALB3T3 cell, Sp-2/O cell, etc. Among them, CHO cell, CHO(dhfrxe2x88x92) cell, 293 cell, etc. are preferred.
Depending on host cells used, transformation is carried out using standard techniques appropriate to such cells.
Transformation of Escherichia species can be carried out in accordance with methods as disclosed in, for example, Proceedings of the National Academy of Sciences of the United State of America, Vol. 69, 2110 (1972), and Gene, Vol. 17, 107 (1982), etc.
Transformation of Bacillus species can be carried out in accordance with methods as disclosed in, for example, Molecular and General Genetics, Vol. 168, 111 (1979), etc.
Transformation of yeast cells can be carried out in accordance with methods as disclosed in, for example, Methods in Enzymology, 194, 182-187(1991), etc.
Transformation of insect cells or insects can be carried out in accordance with methods as disclosed in, for example, Bio/Technology, 6, 47-55, (1988).
Transformation of animal cells can be carried out by methods as disclosed in, for example, Cell Engineering, separate vol. 8, New Cell Engineering Experiment Protocol, 263-267(1995) (Shujun Company), Virology, Vol. 52, 456 (1973), etc.
In introducing the expression vector into cells, known methods such as a calcium phosphate method (Graham, F. L. and van der Eb, A. J.: Virology, 52, 456-467(1973)), an electroporation (Neumann, E. et al., EMBO Journal, 1,841-845(1982)), etc. may be used.
The transformants or transfectants wherein the expression vector carrying the DNA coding for the protein can be obtained according to the afore-mentioned techniques.
Examples of methods for expressing the protein of the present invention stably using animal cells are a method for selecting the cells wherein the above-mentioned expression vector is integrated on the chromosome by means of clone selection. Briefly, the transformant is first selected using the above-mentioned selective marker as an index for selection.
Then the animal cell obtained as such using the selective marker is repeatedly subjected to a clone selection to establish an animal cell strain which stably exhibits a high ability of expressing the protein of the present invention. When a dhfr gene is used as a selective marker, the resistant cells are selected from a culture with a sequentially increased MTX concentration to amplify the DNA coding for the protein of the present invention with dhfr gene in the cells whereby an animal cell strain exhibiting far higher expression can be obtained.
The protein of the present invention or a salt thereof can be also manufactured by culturing the transformant under a condition where the DNA coding for the protein of the present invention can be expressed to express and accumulate the protein of the present invention.
Culture of the transformants (transfectants) of Escherichia or Bacillus species can be carried out preferably in a liquid culture medium. The culture medium may contain carbon sources, nitrogen sources, minerals, etc. which are necessary for growing the transformants. The carbon sources may include glucose, dextrin, soluble starch, sucrose, etc. The nitrogen sources may include organic or inorganic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extracts, bean-cakes, potato extracts, etc. Examples of the minerals may include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc. It is further allowable to add yeast extracts, vitamines, growth-promoting factors, etc. It is suitable that the pH of culture medium is at about 5 to 8.
The culture medium for Escherichia species is, for example, preferably M9 medium which contains glucose and casamino acids (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York, (1972). If necessary, drugs such as 3xcex2-indolyl acrylic acid can be added to the medium to improve efficiency of the promoter. In the case of Escherichia species as a host, the culture is carried out usually at about 15 to 43xc2x0 C. for about 3 to 24 hours. When necessary, aeration and stirring may be applied. In the case of Bacillus species as a host, the culture is carried out usually at about 30 to 40xc2x0 C. for about 6 to 24 hours. When necessary, aeration and stirring may also be applied.
In the case of yeast transformant cells, the culture medium used may include, for example, Burkholder minimum medium (Bostian, K. L. et al., Proceedings of the National Academy of Sciences of the United State of America, Vol. 77, 4505 (1980)), SD medium containing 0.5% casamino acid (Bitter, G. A. et al., Proceedings of the National Academy of Sciences of the United State of America, Vol. 81, 5330 (1984)), etc. It is preferable that the pH of the culture medium is adjusted to be from about 5 to 8. The culture is carried out usually at about 20 to 35xc2x0 C. for about 24 to 72 hours. When necessary, aeration and stirring may be applied.
In the case of the transformants (or transfectants) of insect cells or insects, the culture medium used may include the Grace""s insect medium supplemented with additives such as inactivated 10% bovine serum (Grace, T.C.C., Nature, 195, 788 (1962)). It is preferable that the pH of the culture medium is adjusted to be about 6.2 to 6.4. The culture is usually carried out at about 27xc2x0 C. for about 3 to 5 days. When necessary, aeration and stirring may be applied.
In the case of the transformants (or transfectants) of animal cells, the culture medium used may include MEM medium (Science, Vol. 122, 501 (1952)), DMEM medium (Virology, Vol. 8, 396 (1959)), RPMI 1640 medium (Journal of the American Medical Association, Vol. 199, 519 (1967)), 199 medium (Proceedings of the Society of the Biological Medicine, Vol. 73, 1 (1950)), etc. which are containing, for example, about 5 to 20% of fetal calf serum. It is preferable that the pH is from about 6 to 8. The culture is usually carried out at about 30 to 40xc2x0 C. for about 15 to 60 hours. When necessary, medium exchange, aeration and stirring may be applied. Especially when CHO (dhfrxe2x88x92) cells and dhfr selective marker gene are used, it is preferred to use DMEM medium containing dialyzed fetal bovine serum which rarely contains thymidine.
Separation and purification of the protein from the above-mentioned cultures can be carried out according to the methods described herein below.
To extract the protein from the cultured medium, microorganisms, insects cells or animal cells are collected by known methods after the culture, suspended in a suitable buffer solution, disrupted by sonication, lysozyme treatment and/or freezing and thawing, etc. and, then, a crude protein extract is obtained by centrifugation or filtration. Other conventional extraction or isolation methods can be applied. The buffer solution may contain a protein-denaturing agent such as urea or guanidine hydrochloride or a surfactant such as Triton X-100(trademark).
In the case where proteins are secreted into culture media, supernatants are separated from the microorganisms, insect cells or animal cells after culture and collected by known methods. The culture supernatant containing the protein can be purified by suitable combinations of known methods for separation, isolation and purification. The known methods of separation, isolation and purification may include methods which utilizes a difference in solubility, such as salting out or sedimentation with solvents, methods which utilizes chiefly a difference in the molecular size or weight, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in the electric charge, such as ion-exchange chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing a difference in the hydrophobic property, such as reversed-phase high-performance liquid chromatography, and methods utilizing a difference in the isoelectric point such as isoelectric electrophoresis, etc.
In cases where the protein thus obtained is in a free form, the free-form protein can be converted to a salt thereof by known methods or method analogous thereto. In case where the protein thus obtained is in a salt form vice versa, the protein salt can be converted to a free form or to another salt by known methods or method analogous thereto.
The protein produced by the transformant can be arbitrarily modified or a polypeptide can be partly removed therefrom, by a suitable protein-modifying enzyme before or after the purification. The protein-modifying enzyme may include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase, etc. The amount of the protein of the present invention thus obtained can be measured by a binding assay with a labeled ligand or by an enzyme immunoassays (enzyme linked immunoassays) using specific antibodies.
The antibodies against the protein, the precursor protein, or the partial peptide of the present invention, or a salt thereof are any antibodies such as polyclonal antibodies and monoclonal antibodies which can recognize the protein, the precursor protein, or the partial peptide of the present invention, or a salt thereof (hereinafter referred to as the protein of the present invention).
The antibodies against the protein of the present invention may be manufactured by methods per se known to those of skill in the art or methods similar thereto, using the protein of the present invention as antigen. For example, monoclonal antibodies and/or polyclonal antibodies can be manufactured by the method as given below.
Preparation of Monoclonal Antibody
(a) Preparation of Monoclonal Antibody-Producing Cells
The protein of the present invention is administered to warm-blooded animals either solely or together with a carrier or a diluent to the site favorable for antibody production. In order to potentiate the antibody productivity upon the administration, complete Freund""s adjuvant or incomplete Freund""s adjuvant may be administered. The administration is usually carried out once every 2 to 6 weeks and 2 to 10 times in total. Examples of the applicable warm-blooded animals are monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and fowls. The use of mice or rats is preferred.
In establishing cells which produce monoclonal antibodies, an animal with the detectable antibody titer is selected from animals (e.g. mice) immunized with antigens, then spleen or lymph node is collected after 2 to 5 days from the final immunization and antibody-producing cells contained therein are fused with myeloma cells derived from, homogeneous or heterogeneous animals to obtain monoclonal antibody-producing hybridomas. Measurement of the antibody titer in antisera may, for example, be carried out by reacting a labeled protein, which will be mentioned later, with the antiserum followed by measuring the binding activity of the labeling agent with the antibody. The cell fusion may be carried out, for example, by the method of Koehler and Milstein (Nature, 256, 495, 1975). Examples of the fusion accelerator are polyethylene glycol (PEG), Sendai virus, etc. and the use of PEG is preferred.
Examples of the myeloma cells are those derived from warm-blooded animals such as NS-1, P3U1, SP2/0, AP-1, etc. and the use of P3U1 is preferred. The preferred fusion ratio of the numbers of antibody-producing cells used (spleen cells) to the numbers of myeloma cells is within a range of about 1:1 to 20:1. When PEG (preferably, PEG 1000 to PEG 6000) is added at a concentration of about 10 to 80% followed by incubating at 20 to 40xc2x0 C., preferably, at 30 to 37xc2x0 C., for 1 to 10 minutes, an efficient cell fusion can be carried out.
Various methods may be applied for screening a hybridoma which produces a monoclonal antibody. For example, a supernatant of hybridoma culture is added to a solid phase (e.g. microplate) to which the protein antigen is adsorbed either directly or with a carrier, then anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used when the cells used for the cell fusion are those of mouse) which is labeled with a radioactive substance, an enzyme or the like, or protein A is added thereto and then monoclonal antibodies bound on the solid phase are detected; or a supernatant of the hybridoma culture is added to the solid phase to which anti-immunoglobulin or protein A is adsorbed, and then the protein labeled with a radioactive substance or an enzyme is added and monoclonal antibodies bound with the solid phase is detected.
Selection and cloning of the monoclonal antibody-producing hybridoma may be carried out by methods per se known to those of skill in the art or methods similar thereto. Usually, it is carried out in a medium for animal cells, containing HAT (hypoxanthine, aminopterin and thymidine). With respect to a medium for the selection, for the cloning and for the growth, any medium may be used so far as hybridoma is able to grow therein. Examples of the medium are an RPMI 1640 medium (Dainippon Pharmaceutical Co., Ltd., Japan) containing 1 to 20% (preferably 10 to 20%) of fetal calf serum (FCS), GIT medium (Wako Pure Chemical, Japan) containing 1 to 20% of fetal calf serum and a suitable serum-free medium for hybridoma (SFM-101; Nissui Seiyaku, Japan). The culture temperature is usually 20 to 40xc2x0 C. and, preferably, about 37xc2x0 C. The culture period is usually from five days to three weeks and, preferably, one to two weeks. The culture is usually carried out in 5% carbon dioxide gas. The antibody titer of the supernatant of the hybridoma culture may be measured by the same manner as in the above-mentioned measurement of the antibody titer in the antiserum.
(b) Purification of the Monoclonal Antibody
The separation and purification of the monoclonal antibody may be carried out by methods for separating/purifying immunoglobulin such as salting-out, precipitation with alcohol, isoelectric precipitation, electrophoresis, adsorption/deadsorption using ion exchangers such as DEAE, ultracentrifugation, gel filtration, specific purifying methods in which only an antibody is collected by treatment with an active adsorbent such as an antigen-binding solid phase, protein A or protein G and the bond is dissociated whereupon the antibody is obtained.
Preparation of Polyclonal Antibody
The polyclonal antibody of the present invention can be produced by per se known methods or methods analogous thereto. The method comprises preparing an immunogen (antigen protein) per se or a conjugate of an immunogen with a carrier protein, immunizing a warm-blooded animal in the same manner as described for the production of the monoclonal antibody, harvesting a fraction containing the antibody against the protein of the present invention from the immunized animal, and purifying the harvested antibody.
Referring to the immunogen-carrier protein conjugate for use in the immunization of a warm-blooded animal, the kind of carrier protein and the ratio of the carrier and hapten are not particularly restricted only if the production of the antibody against the hapten conjugated with the particular carrier protein and used for immunization proceeds efficiently. Thus, for example, bovine serum albumin, bovine thyroglobulin, hemocyanine, or the like is coupled in the weight ratio of about 0.1 to 20, preferably about 1 to about 5, to unity of the hapten.
A variety of condensing agents can be used for this coupling between the hapten and the carrier. Thus, for example, a glutaraldehyde, carbodiimide, maleimide, or a thiol or dithiopyridyl group-containing active ester reagent can be employed.
The condensation reaction product is administered to a warm-blooded animal at a site favorable for antibody production, either as it is alone or together with a carrier or diluent. Enhancing antibody production, complete Freund""s adjuvant or incomplete Freund""s adjuvant may be administered. Administration is carried out generally once in about 2 to 6 weeks for a total of about 3 to 10 times.
The polyclonal antibody can be harvested from the blood, ascites fluid, or other body fluid, preferably from the blood, of the host warm-blooded animal.
The polyclonal antibody titer in the antiserum can be determined in the same manner as the determination of monoclonal antibody described hereinbefore. The separation and purification of the polyclonal antibody can be carried out by the same method as that described for the separation and purification of monoclonal antibody.
The antisense DNA having a nucleotide sequence complementary or substantially complementary to the DNA coding for the protein, the precursor protein or the partial peptide of the present invention (hereinafter referred to as the DNA of the present invention) can be any antisense DNA having a nucleotide sequence complementary or substantially complementary to that of the DNA of the present invention and capable of suppressing expression of the DNA.
The nucleotide sequence substantially complementary to the DNA of the present invention may, for example, be a nucleotide sequence having an identity of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, and for still better results, not less than about 95% to the total nucleotide sequence or partial nucleotide sequence of the nucleotide sequence complementary to that the DNA of the present invention. Particularly preferred is an antisense DNA having an identity of not less than about 70%, preferably not less than about 80%, and more preferably not less than about 90%, and for still better results, not less than about 95% to the nucleotide sequence of the domain, of the complete nucleotide sequence complementary to that of the DNA of the present invention, which encodes the N-terminal region of the protein of the present invention (e.g. the nucleotide sequence of the domain around the initiation codon). The antisense DNA can be synthesized using a known DNA synthesis hardware.
The protein, the partial peptide or a salt thereof of the present invention has activities such as a lecithin-cholesterol acyltransferase-like activity and so on, and transfers the xcex2-acyl group (fatty acid) of lecithin (phosphatidylcholine) to the 3xcex2-OH group of cholesterol, so that it consumes the equivalent moles of lecithin and unesterified cholesterol and produces the equivalent moles of cholesteryl ester and lysolecithin.
Uses for the protein, the precursor protein, the partial peptide or a salt thereof (hereinafter sometimes referred to collectively as the protein, etc. of the present invention), the DNA coding for the protein of the present invention (hereinafter sometimes referred to briefly as the DNA of the present invention), the antibody against the protein, the precursor protein, the partial peptide or a salt thereof of the present invention (hereinafter sometimes referred to as the antibody of the present invention), and the antisense DNA of the present invention are now described.
(1) Medicinal Products Such as Drugs for Treating or Preventing Various Diseases
As LCAT concerns a cholesterol metabolism, when there is a mutation or deletion on the DNA coding for the LCAT, or a degree of LCAT is decreasing, various diseases (e.g. arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity or hypertriglyceridemia) are arisen from. Therefore, the protein of the present invention etc., and the DNA of the present invention is used for a pharmaceutical agent for treating or preventing arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity, hypertriglyceridemia.
Moreover, the protein of the present invention having LCAT-like activity can be used as a drug for treating or preventing arteriosclerosis, atherosclerosis, hyperlipidemia, obesity, inflammatory diseases, senescence, diseases of brain, and renal disorder.
For example, when there is a patient who is not able to metabolize cholesterol sufficiently or normally in the cell because of a decrease or a defect of LCAT, the role-of the protein of the present invention for said patient can be expected sufficiently or normally by:
(a) administering the DNA coding for the protein, etc. of the present invention to the patient to express it;
(b) inserting the DNA coding for the protein, etc. of the present invention into cells to express it and transplanting the cells to said patient, or (c) administering the protein, etc. of the present invention to the patient.
When the DNA of the present invention is used as the above-mentioned pharmaceutical agent, said DNA may be used alone or after inserting it into a suitable vector such as retrovirus vector, adenovirus vector, adenovirus-associatedvirus vector, pox virus etc. followed by subjecting the product vector to a conventional means. The DNA can also be administered as xe2x80x9cnakedxe2x80x9d DNA, with physiologically acceptable carriers such as adjuvants toassist in uptake, by xe2x80x9cgenexe2x80x9d gun or by a catheter such as a catheter with a hydrogel.
If one wishes to use the protein, etc. of the present invention, one would use it in a purified form, preferably in a purity of at least 90%, more preferably at least 95%, still more preferably at least 98% and most preferably at least 99%.
For example, the protein, etc. of the present invention can be used orally in the form of tablets which may be sugar coated if necessary, capsules, elixirs, microcapsules etc., or non-orally in the form of injectable preparations such as aseptic solutions and suspensions in water or other pharmaceutically acceptable liquids. These preparations can be produced by mixing the protein, etc. of the present invention with physiologically acceptable carriers, flavoring agents, excipients, vehicles, antiseptics, stabilizers, binders etc. in unit dosage forms required for generally accepted manners of pharmaceutical preparation. Active ingredient contents in these preparations are set so that an appropriate dose within the specified range is obtained.
Additives which can be mixed in tablets, capsules etc. include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid, lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose and saccharin, and flavoring agents such as peppermint, akamono oil and cherry. When the unit dosage form is the capsule, the above-mentioned materials may further incorporate liquid carriers such as oils and fats. Sterile compositions for injection can be formulated by ordinary methods of pharmaceutical preparation such as by dissolving or suspending active ingredients, naturally occuring vegetable oils such as sesame oil and coconut oil, etc. in vehicles such as water for injection to create pharmaceutical compositions.
Aqueous liquids for injection include physiological saline and isotonic solutions containing glucose and other auxiliary agents, e.g., D-sorbitol, D-mannitol and sodium chloride, and may be used in combination with appropriate dissolution aids such as alcohols, e.g., ethanol, polyalcohols, e.g., propylene glycol and polyethylene glycol, nonionic surfactants, e.g., polysorbate 80(trademark) and HCO-50 etc. Oily liquids include sesame oil and soybean oil, and may be used in combination with dissolution aids such as benzyl benzoate and benzyl alcohol. Furthermore the above-mentioned materials may also be formulated with buffers, e.g., phosphate buffer and sodium acetate buffer; soothing agents, e.g., benzalkonium chloride, procaine hydrochloride; stabilizers, e.g., human serum albumin, polyethylene glycol; preservatives, e.g., benzyl alcohol, phenol; antioxidants etc. Normally, an appropriate ampule is filled in with the thus-prepared pharmaceutical composition such as an injectable liquid.
The vector comprising the DNA of the present invention can be formulated as well as mentioned above, and usually can be used non-orally.
Because the thus-obtained preparation is safe and of low toxicity, it can be administered to humans or mammals (e.g., rat, mouse, guinia pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, etc.).
The dose of the protein, etc. of the present invention may vary depending on subject disease, subject of administration, way of administration, and so on. When the protein, etc. of the present invention is used, for example, for treating hyperlipidemia by oral administration, the dose of the protein, etc. of the present invention is normally about 0.1 to 100 mg, preferably 1.0 to 50 mg, and more preferably 1.0 to 20 mg per day for an adult human (weighing 60 kg). When the protein, etc. of the present invention is used, for example, for treating hyperlipidemia by non-oral administration, it is advantageous to administer the protein, etc. of the present invention in the form of injectable preparation at a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per administration by an intravenous injection for an adult human (weighing 60 kg), depending on subject of administration, subject disease and so on. For other animal species, corresponding does as converted per 60 kg weight can be administered.
(2) Screening for Candidate Medicinal Compounds
Since the protein etc. of the present invention has LCAT-like activity, any compound or salt that stimulates the function (e.g. LCAT activity) of the protein etc. of the invention can be used as a pharmaceutical preparation such as a drug for treating or preventing arteriosclerosis, atherosclerosis, hyperlipidemia, atherosclerosis hypercalorism, obesity, hypertriglyceridemia, inflammatory diseases, senescence, diseases of brain, renal disorder and other diseases.
Among them, the protein etc. of the present invention can be used for a drug for treating on preventing arteriosclerosis, atherosclerosis, hyperlipidemia, obesity, inflammatory diseases, senescence, diseases of brain, and renal disorder.
On the other hand, any compound or salt that inhibits the function of the protein etc. of the invention can also be used as a pharmaceutical preparation such as a drug for treating or preventing malnutrition, abetalipoproteinemia, inflammatory diseases, Tangier disease (analphalipoproteinemia), and other diseases.
Therefore, the protein etc. of the present invention is of value as a screening reagent for those compounds and salts which activate or inhibit the function of the protein or equivalent of the present invention.
The present invention, therefore, provides
(1) a screening method for those compounds or salt thereof which stimulate the function (e.g. LCAT-like activity) of the protein, the partial peptide, or a salt thereof of the present invention (hereinafter sometimes referred to briefly as an agonist) or those compounds or a salt thereof which inhibit the function of the protein, the partial peptide or a salt thereof of the present invention (hereinafter sometimes referred to briefly as an antagonist), which comprises using the protein, the partial peptide, or a salt thereof of the invention.
More specifically, the present invention provides
(2) a screening method for said agonist or antagonist, which comprises comparing the case (i) in which the protein, the partial peptide, or a salt thereof of the invention is exposed to lecithin and unesterified cholesterol with (ii) the case in which the protein, the partial peptide, or a salt thereof of the invention is exposed to lecithin, unesterified cholesterol, and a test compound.
Typically, this screening method comprises assaying the LCAT-like activity of the protein etc. of the invention in the above-mentioned cases (i) and (ii) and comparing the results.
For use in the present invention, lecithin may for example be a commercial egg white lecithin (e.g. Sigma).
For use in the present invention, unesterified cholesterol may for example be a [14C]-labeled cholesterol (e.g. Amersham).
The screening method according to the present invention is preferably practiced using a proteoliposomal preparation containing such lecithin and unesterified cholesterol. This proteoliposomal preparation contains Apo A-1, [14C]-cholesterol, and egg white lecithin in a molar ratio of 0.8:12.5:250.
The test compound that can be used includes but is not limited to peptides, proteins, nonpeptides, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts. Those compounds may be novel compounds or known compounds.
For use in the above screening method, the protein etc. of the present invention is suspended in a suitable screening buffer to prepare a sample of the protein etc. of the invention. The buffer mentioned above may be any buffer solution that does not interfere with the reaction of the protein etc. of the present invention with lecithin and unesterified cholesterol, thus including phosphate buffer and Tris-HCl buffer within the pH range of about 4-10 (preferably pH 6-8).
The LCAT activity of the protein etc. of the present invention can be assayed by the method described in Lipoprotein Analysis (Converse, C. A. and Skinner, R. E. (eds), 1992, IRL Press, Oxford, the chapter authored by Gillet, M. P. T. and Owen, J. S. (pp. 187-201), either as such or as modified.
When a test compound enhanced LCAT activity in case (ii) by not less than about 20%, preferably not less than 30%, more preferably not less than 50%, as compared with case (i), the particular compound can be adopted as an agonist of the LCAT activity of the protein etc. of the invention.
Conversely, when a test compound inhibited LCAT activity in case (ii) by not less than about 20%, preferably not less than about 30%, more preferably not less than about 50%, as compared with case (i), the particular compound can be adopted as an antagonist of the LCAT activity of the protein etc. of the present invention.
The screening kit according to the present invention includes the protein of the invention, a precursor protein or fragment peptide thereof, or a salt of any of them. A typical screening kit according to the present invention is as follows.
[Screening Reagents]
(1) Screening buffer
Tris-HCl buffer (pH 7.4), human serum albumin
(2) Protein sample
The protein, precursor protein, fragment peptide, or salt according to the present invention
(3) Proteoliposomal preparation
[14C]-cholesterol (105 cpm/ml)/Apo A1/[14C]cholesterol/egg white lecithin (molar proportions: 0.8:12.5:250)
(4) Detection
Thin-layer chromatography
[Method]
LCAT activity can be assayed by using said solution of the present protein etc. of the invention and said proteoliposomal preparation. Typically, the proteoliposomal preparation can be prepared by mixing Apo A-1, [14C]-cholesterol, and egg white lecithin in the molar proportions of 0.8:12.5:250 and incubating the mixture at 37xc2x0 C. for 30 minutes.
[Protocol]
Incubate the protein solution, the proteoliposomal preparation, and a test compound at 37xc2x0 C. for 1 hour, isolate the product cholesteryl ester by thin-layer chromatography using hexane/diethyl ether/acetic acid (83:16:1) as developer, and assay. The results can be expressed in nanomoles of free cholesterol esterified per hour.
The compound or salt obtained according to the above screening protocol or by using the above-mentioned screening kit of the invention is a compound (e.g. a member of the class consisting of peptides, proteins, nonpeptides, synthetic compounds,. fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc.) which either activates or inhibits the function (e.g. LCAT-like activity) of the protein etc. of the present invention.
As the salt of such a compound, the same kind of salt as the salt of the protein of the invention, mentioned-above can be used.
The compound which activates the function (e.g. LCAT-like activity) of the protein etc. of the present invention can be used as a drug, e.g. a drug for treating or preventing arteriosclerosis, atherosclerosis, atherosclerosis hyperlipidemia, hypercalorism, obesity, hypertriglyceridemia, inflammatory diseases, senescence, diseases of brain, renal disorder and other diseases.
Among them the protein etc. of the present invention can be used for a drug for treating on preventing arteriosclerosis, atherosclerosis, hyperlipidemia, obesity, senescence, diseases of brain, and renal disorder.
On the other hand, the compound which inhibits the function of the protein or equivalent of the invention can be used as a drug, e.g. a drug for treating or preventing malnutrition, abetalipoproteinemia, inflammatory diseases, Tangier disease, and other diseases.
When the compound obtained by the screening method of the present invention or by using the screening kit of the present invention is used as agent for treating or preventing the diseases mentioned above, it can be formulated to tablets, capsules, slixirs, microcapsules, aspetic solution, suspensions or the like in the same way as the pharmaceutical composition comprising the protein, etc. of the present invention as mentioned above.
Because the thus-obtained preparation is safe and of low toxicity, it can be administered to humans or mammals (e.g., rat, mouse, guinia pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, etc.).
The dose of the protein, etc. of the present invention may vary depending on subject disease, subject of administration, and so on. When the compound which promotes the function of the protein of the present invention (e.g. LCAT-like activity, etc.) is used, for example, for treating hyperlipidemia by oral administration, the dose of the compound is normally about 0.1 to 100 mg, preferably 1.0 to 50 mg, and more preferably 1.0 to 20 mg per day for an adult human (weighing 60 kg). When the compound which promotes the function of the protein of the present invention (e.g. LCAT-like activity, etc.) is used, for example, for treating hyperlipidemia by non-oral administration, it is advantageous to administer the compound in the form of injectable preparation at a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per administration by an intravenous injection for an adult human (weighing 60 kg), depending on subject of administration, subject disease and so on. For other animal species, corresponding doses as converted per 60 kg weight can be administered.
When the compound which inhibits the function of the protein of the present invention (e.g. LCAT-like activity, etc.) is used, for example, for treating abetalipoproteinemia by oral administration, the dose of the compound is normally about 0.1 to 100 mg, preferably 1.0 to 50 mg, and more preferably 1.0 to 20 mg per day for an adult human (weighing 60 kg). When the compound which inhibits the function of the protein of the present invention (e.g. LCAT-like activity, etc.) is used, for example, for treating abetalipoproteinemia by non-oral administration, it is advantageous to administer the compound in the form of injectable preparation at a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per administration by an intravenous injection for an adult human (weighing 60 kg), depending on subject of administration, subject disease and so on. For other animal species, corresponding does as converted per 60 kg weight can be administered.
(3) Quantitative Determination of the Protein of the Present Invention
The antibody of the present invention is capable of specifically recognizing the protein, etc. of the present invention and, accordingly, it can be used for quantitative determination of the protein, etc. of the present invention in test liquid samples and particularly for quantitative determination by sandwich immunoassays.
Thus, the present invention provides, for example, the following methods:
(i) a quantitative determination of the protein, etc. of the present invention in a test liquid sample, which comprises
(a) competitively reacting the test liquid sample and a labeled protein, etc. of the present invention with the antibody of the present invention, and
(b) measuring the ratio of the labeled protein, etc. of the present invention binding with said antibody; and
(ii) a quantitative determination of the protein, etc. of the present invention in a test liquid sample, which comprises
(a) reacting the test liquid sample with an antibody immobilized on an insoluble carrier and a labeled antibody simultaneously or continuously, and (b) measuring the activity of the labeling agent on the insoluble carrier, wherein one antibody is capable of recognizing the N-terminal region of the protein, etc. of the present invention while another antibody is capable of recognizing the C-terminal region of the protein, etc. of the present invention.
When the monoclonal antibody of the present invention recognizing a protein, etc. of the present invention (hereinafter, sometimes referred to as xe2x80x9cmonoclonal antibody of the present inventionxe2x80x9d) is used, the quantity of the protein, etc. of the present invention can be measured and, moreover, the protein, etc. of the present invention can be detected by means of a tissue staining, etc. as well. For such an object, antibody molecules per se may be used, or F(abxe2x80x2)2 Fabxe2x80x2 or Fab fractions of the antibody molecule may also be used.
There is no particular limitation for the measuring method using the antibody of the present invention and any measuring method may be used so far as it relates to a method in which the amount of antibody, antigen or antibody-antigen complex, depending on or corresponding to the amount of antigen, e.g. the amount of the protein, etc. of the present invention in the liquid sample to be measured, is detected by a chemical or a physical means and then calculated using a standard curve prepared by a standard solution containing the known amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are suitably used and, in terms of sensitivity and specificity, the sandwich method which will be described herein later is particularly preferred.
Examples of the labeling agent used in the measuring method using the labeling substance are radioisotopes, enzymes, fluorescent substances, luminescent substances, colloids, magnetic substances, etc. Examples of the radioisotope are [125I], [131I], [3H] and [14C]. Preferred examples of the enzyme are those which are stable and with much specific activity, such as xcex2-galactosidase, xcex2-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase. Examples of the fluorescent substance are fluorescamine, fluorescein isothiocyanate, etc. Examples of the luminescent substance are luminol, luminol derivatives, luciferin, lucigenin, etc. Further, a biotin-avidin system may also be used for binding an antibody or antigen with a labeling agent.
In insolubilization (immobilization) of antigens or antibodies, a physical adsorption may be used or a chemical binding which is usually used for insolubilization or immobilization of proteins or enzymes may be used as well. Examples of the carrier are insoluble polysaccharides such as agarose, dextran and cellulose; synthetic resins such as polystyrene, polyacrylamide and silicone; glass; etc.
In a sandwich method, the test liquid is allowed to react with an insolubilized monoclonal antibody of the present invention (the first reaction), then it is allowed to react with another labeled monoclonal antibody of the present invention (the second reaction) and the activity of the labeling agent on the insoluble carrier is measured whereupon the amount of the protein, etc. of the present invention in the test liquid can be determined. The first reaction and the second reaction may be conducted reversibly or simultaneously or they may be conducted with an interval. The type of the labeling agent and the method of insolubilization may be the same as those mentioned hereinbefore. In the immunoassay by means of a sandwich method, it is not always necessary that the antibody used for the labeled antibody and the antibody for the solid phase is one type or one species but, with an object of improving the measuring sensitivity, etc., a combination of two or more antibodies may be used as well.
In the method of measuring the protein, etc. of the present invention by the sandwich method of the present invention, the preferred monoclonal antibodies of the present invention used for the first and the second reactions are antibodies wherein their sites binding to the protein of the present invention are different from each other. Thus, antibodies used in the first and the second reactions are those wherein, when an antibody used in the second reaction recognizes the C-terminal region of the protein, etc. of the present invention, then another antibody recognizing the site other than C-terminal regions, e.g. recognizing the N-terminal region, is preferably used in the first reaction.
The monoclonal antibody of the present invention may be used in a measuring system other than the sandwich method such as a competitive method, an immunometric method and a nephrometry.
In the competitive method, an antigen in the test solution and a labeled antigen are allowed to react with an antibody in a competitive manner, then an unreacted labeled antigen (F) and a labeled antigen (B) binding with an antibody are separated (i.e. B/F separation) and the labeled amount of any of B and F is measured whereupon the amount of the antigen in the test solution is determined. With respect to the method for such a reaction, there are a liquid phase method in which a soluble antibody is used as the antibody and the B/F separation is conducted by polyethylene glycol, a second antibody to the above-mentioned antibody, etc.; and a solid phase method in which an immobilized antibody is used as the first antibody or a soluble antibody is used as the first antibody while an immobilized antibody is used as the second antibody.
In the immunometric method, an antigen in the test solution and an immobilized antigen are subjected to a competitive reaction with a certain amount of a labeled antibody followed by separation into solid and liquid phases or the antigen in the test solution and an excess amount of labeled antibody are allowed to react, then an immobilized antigen is added to bind an unreacted labeled antibody with the solid phase and separated into solid and liquid phases. After that, the labeled amount of any of the phases is measured to determine the antigen amount in the test solution.
In the nephrometry, the amount of insoluble sediment which is produced as a result of the antigen-antibody reaction in a gel or in a solution is measured. Even when the antigen amount in the test solution is small and only a small amount of the sediment is obtained, a laser nephrometry wherein scattering of laser is utilized can be suitably used.
In applying each of those immunological measuring methods (immunoassays) to the measuring method of the present invention, it is not necessary to set up any special condition, operation, etc. therefor. A measuring system (assay system) for the protein of the present invention may be constructed taking the technical consideration of the persons skilled in the art into consideration in the conventional conditions and operations for each of the methods. With details of those conventional technical means, a variety of reviews, reference books, etc. may be referred to.
They are, for example, Hiroshi Irie (ed): xe2x80x9cRadioimmunoassayxe2x80x9d (Kodansha, Japan, 1974); Hiroshi Irie (ed): xe2x80x9cRadioimmunoassay; Second Seriesxe2x80x9d (Kodansha, Japan, 1979); Eiji Ishikawa et al. (ed): xe2x80x9cEnzyme Immunoassayxe2x80x9d (Igaku Shoin, Japan, 1978); Eiji Ishikawa et al. (ed): xe2x80x9cEnzyme Immunoassayxe2x80x9d (Second Edition) (Igaku Shoin, Japan, 1982); Eiji Ishikawa et al. (ed): xe2x80x9cEnzyme Immunoassayxe2x80x9d (Third Edition) (Igaku Shoin, Japan, 1987); xe2x80x9cMethods in Enzymologyxe2x80x9d Vol. 70 (Immunochemical Techniques (Part A)); ibid. Vo. 73 (Immunochemical Techniques (Part B)); ibid. Vo. 74 (Immunochemical Techniques (Part C)); ibid. Vo. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)); ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)); ibid. Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (Academic Press); etc.
By using the antibody of the present invention in the above manner, the protein of the present invention can be assayed with high sensitivity.
In addition, when decrease in concentration of the protein, etc. of the present invention is detected by determining the concentration of the protein, etc. of the present invention by using the antibody against the protein of the present invention, it may lead, with high probability, to the diagnosis of various diseases such as arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity, hypertriglyceridemia, inflammatory diseases, senescence, diseases of brain, renal disorder and so on.
When increase in concentration of the protein, etc. of the present invention is detected, it may lead, with high probability, to the diagnosis of various diseases such as malnutrition, abetalipoproteinemia or Tangier disease and so on.
Thus, the antibody of the present invention is useful as a diagnostic agent for the above-mentioned diseases.
Furthermore, the antibody of the present invention can be used for the purpose of detecting the protein of the present invention which may be present in test samples such as body fluids or tissues. The antibody can also be used for the construction of an antibody column for purification of the protein of the present invention, detection of the protein of the present invention in the fractions in the course of purification, and analysis of the behavior of the protein of the present invention in the test cell.
(4) Gene Diagnostic Agent
By using the DNA of the present invention as a probe, for instance, an abnormality (gene abnormality) of the DNA or mRNA coding for the protein of the present invention or its partial peptide in humans or mammals (e.g. rat, mouse, guinea pig, rabbit, sheep, swine, bovine, horse, cat, dog, monkey, chimpanzee, etc.) can be detected. Therefore, the DNA of the present invention is useful as a gene diagnostic agent for the damage to the DNA or mRNA, mutation thereof, or decreased expression thereof, or increased expression or over expression of the DNA or mRNA.
For example, when the increase of the mRNA coding for the protein, etc. or the increase of the protein, etc. of the present invention is detected, it may be lead to the diagnosis of malnutrition, abetalipoproteinemia or Tangier disease, etc.
On the other hand, the deficit or lack of the DNA or mRNA or the decrease of the protein, or a mutation of the DNA is detected by the PCR-SSCP assay etc. is detected, it may be lead to the diagnosis of arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity, hypertriglyceridemia, inflammatory diseases, senescence, diseases of brain, renal disorder, etc.
The above-mentioned gene diagnosis using the DNA of the present invention can be carried out by, for example, the per se known Northern hybridization assay or PCR-SSCP assay (Genomics, 5, 874-879 (1989); Proceedings of the National Academy of Sciences of the United States of America, 86, 2766-2770 (1989)).
When increase in expression of the mRNA coding for the protein, etc. of the present invention is detected by Northern hybridization assay, it may lead, with high probability, to the diagnosis of malnutrition, abetalipoproteinemia or Tangier disease, etc.
When decrease in expression of the mRNA or a mutation of the DNA is detected by the PCR-SSCP assay is detected it may lead, with high probability, to the diagnosis of arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity, hypertriglyceridemia, etc.
(5) Pharmaceutical Composition Containing the Antisense DNA
The antisense DNA which is capable of complementarily binding to the DNA coding for the protein, etc. of the present invention and suppresses the expression of the DNA and the protein, etc. of the present invention is capable of inhibiting the function of the protein, etc. or the DNA coding for the protein, etc. of the present invention which show the above-mentioned activities in vivo. Therefore, this antisense DNA is used for an agent for treating or preventing various diseases such as malnutrition, abetalipoproteinemia or Tangier disease, etc.
When the antisense DNA is used for the agent for treating or preventing the diseases mentioned above, it can be formulated in the same way as the composition for treating or preventing the disease mentioned above containing the DNA of the present invention.
The DNA may be used alone or after inserting it into a suitable vector such as retrovirus vector, adenovirus vector, adenovirus-associated virus vector, pox virus etc. followed by subjecting the product vector to a conventional means. The DNA can be administered as xe2x80x9cnakedxe2x80x9d DNA, or with physiologically acceptable carriers such as adjuvants to assist in uptake, by xe2x80x9cgenexe2x80x9d gun or by a catheter such as a catheter with a hydrogel.
In addition, this antisense DNA can be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA of the present invention or the status of its expression in various tissues and cells.
(6) Pharmaceutical Compositions Containing the Antibody of the Present Invention
Of the antibody according to the present invention, those species which neutralize the activity of the protein, etc. of the present invention can be used as drugs, such as an agent for treating or preventing diseases such as malnutrition, abetalipoproteinemia or Tangier disease, etc.
The above-mentioned composition for treating or preventing the diseases mentioned above containing the antibody of the present invention can be administered either orally or otherwise to human and other mammals (e.g. rat, rabbit, sheep, swine, cattle, cat, dog, monkey), in the form of an antibody solution as such or in the form of a pharmaceutical composition having an appropriate dosage form.
The dosage is dependent on the recipient, target disease, symptom, administration route, and other factors. Generally, however, for treating or preventing abetalipoproteinemia in a human adult, for instance, the antibody capable of neutralizing the activity of the protein, etc. of the present invention can be administered, by the intravenous route, in a single dose of about 0.01 to 20 mg/kg body weight, preferably 0.1 to 10 mg/kg body weight, or more preferably about 0.1 to 5 mg/kg body weight, about 1 to 5 times a day, or preferably about 1 to 3 times a day. For administration by other routes and for oral administration, the dosage can be selected using the above dosage schedule as a reference. In case of presenting with particularly severe symptoms, the dosage may be increased according to the condition.
The antibody of the present invention which neutralizes the activity of the protein, etc. of the present invention can be administered either as it is or in the form of a suitable pharmaceutical composition. The pharmaceutical composition comprises the antibody or its salt and a pharmaceutically acceptable carrier, diluent, or excipient. The composition can be provided in various dosage forms suited for oral administration or non-oral administration.
The composition for oral administration, for instance, includes solid and liquid dosage forms such as tablets (including dragees, film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrup, emulsion, suspension, etc. Such dosage forms can be manufactured by the per se known procedures and contain a carrier, diluent or excipient which is generally included in pharmaceutical formulations. The carrier or excipient for tablets includes but is not limited to lactose, starch, sucrose, and magnesium stearate.
The composition for non-oral administration may for example be an injectable product or a suppository. The injectable product includes intravenous, subcutaneous, intradermal, intramuscular, drip, and other injections. Such injections can be prepared by the per se known procedures, for example by dissolving, suspending, or emulsifying the antibody or salt in a sterile aqueous or oily vehicle which is generally used in the manufacture of injectable products. The aqueous vehicle for injections includes physiological saline and various isotonic solutions containing glucose and/or the like and may be supplemented with a suitable solubilizer such as alcohols (e.g. ethanol), polyols (e.g. propylene glycol, polyethylene glycol), nonionic surfactants [polysorbate 80, HCO-50 (polyoxyethylene(50 mol)-hydrogenated castor oil adduct)], etc. The oily vehicle includes but is not limited to sesame oil and soybean oil. Benzyl benzoate, benzyl alcohol, etc. may also be used as solubilizers. Injections thus prepared are provided as filled in suitable ampules. Suppositories for rectal administration can be manufactured by mixing said antibody or salt with any of the conventional suppository bases.
The above pharmaceutical composition for oral or non-oral administration can be conveniently provided in unit dosage forms suited for delivery of the unit dose of the active ingredient. The unit dosage form may for example be the above-mentioned tablet, pill, capsule, injection (ampule) or suppository. Preferably, the amount of said antibody or salt per unit dosage form is generally 5-500 mg and preferably 5-100 mg for injectable products or 10-250 mg for other products.
The foregoing composition may contain other active ingredients unless their formulation with said antibody or salt results in unfavorable interactions.
(7) Construction of a Transgenic Animal
The present invention further provides a non-human mammal harboring a foreign DNA coding for the protein of the present invention (hereinafter referred to briefly as foreign DNA) or a mutant thereof (sometimes referred to briefly as a foreign mutant DNA).
Thus, the present invention provides
(1) a non-human mammal harboring a foreign DNA of the present invention or a foreign mutant DNA thereof:
(2) the non-human mammal according to (1) which is a rodent:
(3) the non-human mammalian according to (2) wherein the rodent is a mouse; and
(4) a recombinant vector containing the foreign DNA of the present invention or a foreign mutant DNA thereof and capable of being expressed in a mammal.
The non-human mammal harboring the foreign DNA of the present invention or a foreign mutant DNA thereof (hereinafter referred to briefly as the transgenic animal of the present invention) can be constructed by transferring the objective DNA to a germinal cell such as an unfertilized egg cell, fertilized egg cell, or sperm cell or its primordial cell, preferably in the period of embryogenesis in the ontogenesis of a non-human mammal (more preferably in the stage of a single cell or a fertilized egg cell and generally at the 8-cell stage or earlier), by the calcium phosphate method, electric pulse method, lipofection method, agglutination method, microinjection method, particle gun method, or DEAE-dextran method.
The non-human mammal used includes bovine, swine, sheep, goat, rabbit, canine, feline, guinea pig, hamster, murine, rat, and so on. From the standpoint of construction of a diseased animal model, rodents which have comparatively short ontogenesis and life cycles and can be easily bred, particularly mice (e.g. pure strains such as C57BL/6, DBA2, etc. and hybrid strains such as B6C3F1, BDF1, B6D2F1, BALB/c, ICR, etc.) or rats (e.g. Wistar, SD, etc.) are preferred.
The xe2x80x9cmammalxe2x80x9d as mentioned with reference to the recombinant vector capable of being expressed in a mammal includes the same non-human mammals as those mentioned above and humans.
The mutant DNA includes not only the DNAs available upon variation (e.g. mutation) of the nucleotide sequence of the original DNA of the present invention, for example, upon addition or deletion of nucleotide sequence or substitution of other, and includes abnormal DNAs.
The term xe2x80x9cabnormal DNAxe2x80x9d as used herein means any DNA that causes an expression of an abnormal protein of the present invention, for example, an expression of a protein which suppresses the function of the normal protein of the present invention.
The foreign DNA of the present invention may be one derived from a mammal of the same species as the host animal or a mammal of a different species. For transfer of the DNA of the present invention to the host animal, it is generally advantageous to use a DNA construct prepared by linking the DNA at downstream of a promoter capable of being expressed in animal cells. For example, in transferring the human-derived DNA of the present invention, this human DNA of the present invention can be linked at downstream of a promoter capable of causing expression of DNAs derived from various animals (e.g. rabbit, canine, feline, guinea pig, hamster, rat, murine, etc.) harboring the DNA of the present invention having high homology thereto to prepare a DNA construct (e.g. a vector) which can then be microinjected into the fertilized egg cell of a host mammal such as a fertilized murine egg cell, whereby a transgenic mammal showing a high expression of the DNA of the present invention can be provided.
Examples of the expression vector used for the protein of the present invention are plasmids derived from E. coli, plasmids derived from B. subtilis, plasmids of the yeast origin, xcex phage and other bacteriophages, retroviruses such as Molony leukemia virus, and animal viruses such as vaccinia virus and vaculovirus. Preferable examples are plasmids of the E. coli origin, plasmids of the B. subtilis origin, and yeast-derived plasmids.
The promoter for the regulation of the expression of the DNA are (1) promoters for DNAs derived from viruses (e.g. simian virus, cytomegalovirus, Molony leukemia virus, JC virus, papilloma virus, poliovirus, etc.), (2) promoters derived from mammals (e.g. man, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) for albumin, insulin II, uroprakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelet-derived growth factor xcex1, keratin K1, K10, and K14, collagen type I and type II, cyclic AMP-dependent protein kinase xcex2I subunit, dystrophin, tartaric acid-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium/potassium-exchanging adenosinetriphosphatase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloprotease I tissue inhibitor, MHC Class I antigen (H-2L), H-ras, renin, dopamine xcex2-hydroxylase, thyroid peroxidase (TPO), poly-peptide chain elongation factor 1xcex1 (EF-1xcex1), xcex2actin, xcex1- and xcex2-myosin heavy chain, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin H chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle -xcex1 actin, preproenkephalin A or vasopressin, and so on. Preferable promoters are promoters conducive to high expression in the whole body, such as cytomegalovirus promoter, human polypeptide chain elongation factor 1xcex1 (EF-1xcex1) promoter, and human and chicken xcex2-actin promoters.
The vector preferably has a sequence for terminating the transcription of the objective mRNA (generally called terminator) in the transgenic mammal. The examples of the sequence are sequences derived from viruses, various mammals. Preferable examples are the SV40 terminator derived from simian virus, and so on.
In addition, for enhancing the expression of the objective DNA, it is possible, depending on the specific objective, to link the splicing signal, enhancer domain, a portion of the eucaryotic DNA intron, etc. at upstream of the 5xe2x80x2-end of the promoter region, between the promoter region and the translated region, or at downstream of the 3xe2x80x2-end of the translated region.
The translated region of the normal protein of the present invention can be obtained, as the whole or part of the genomic DNA, from the DNAs derived from the liver, kidney, or thyroid cells or fibroblasts of various mammals (e.g. rabbit, canine, feline, guinea pig, hamster, rat, murine, man, etc.) or from various commercial genomic DNA libraries, or starting with the complementary DNAs prepared from RNAs derived from the liver, kidney, thyroid cells or fibroblasts by the known technique. The foreign abnormal DNA can be constructed by mutating the translated region of the normal protein obtained from the above-mentioned cells or tissues by the mutagenesis method.
The translated region can be prepared as a DNA construct which can be expressed in a transgenic animal, by the routine recombinant DNA technique, i.e. by coupling it at downstream of the promoter and, if desired, at upstream of the transcription termination site.
The transfer of the DNA of the present invention at the fertilized egg cell stage insures that the DNA will be ubiquitous in all the germ cells and somatic cells of the host mammal. The presence of the DNA of the present invention in the germ cells of the transgenic animal following DNA transfer means that all the germ cells and somatic cells of all the progeny of the transgenic animal harbor the DNA of the present invention. Thus, the offspring of animals of this line to which DNA is passed down have the DNA of the present invention in their germ cells and somatic cells.
The non-human mammal to which the foreign normal DNA of the present invention has been transferred can be verified by mating to retain the DNA stably and then bred as a strain harboring the transferred DNA from generation to generation under the usual breeding conditions. The transfer of the DNA of the present invention in the fertilized egg cell stage is carried out in such a manner that the transferred DNA will be present in excess in all the germ cells and somatic cells of the transgenic animal. The presence of an excess of the DNA of the present invention in the germ cells of the transgenic animal means that all the progeny of this line harbor an excess of the DNA of the present invention in their germ cells and somatic cells. By preparing homozygous animals having the transferred DNA in both homologous chromosomes and mating the animals of both sexes, they can be bred serially so that all the progeny may harbor an excess of the DNA.
The non-human mammal harboring the normal DNA of the present invention features a high expression of the DNA and may eventually develop a hyperergasia of the protein of the present invention through activation of the function of the endogenous normal DNA and, therefore, can be utilized as an animal model of the disease. For example, by using the transgenic animal harboring the normal DNA of the present invention, it is possible to study the hyperergasia of the protein of the present invention to elucidate the mechanisms of diseases with which the protein of the present invention is associated, and explore therapeutic modalities for the diseases.
Furthermore, the mammal to which the foreign normal DNA of the present invention has been transferred presents with symptoms due to an increase in the free protein of the present invention and, therefore, can also be used in the screening of therapeutic drugs for diseases with which the protein of the present invention is associated.
On the other hand, the non-human mammal harboring the foreign abnormal DNA of the present invention can be verified by mating to retain the DNA stably and then bred as a line harboring the DNA from generation to generation under the usual breeding conditions.
Moreover, it is possible to incorporate the objective DNA in the above-mentioned plasmid for use as a starting material. The DNA construct with the promoter can be prepared by the routine recombinant DNA technique. Transfer of the abnormal DNA of the present invention in the fertilized egg cell stage insures that the transferred DNA will be ubiquitous in all the germ cells and somatic cells of the host mammal. The presence of the abnormal DNA of the present invention in the germ cells of the transgenic animal means that all the offspring of this transgenic animal harbor the abnormal DNA of the present invention in all of their germ cells and somatic cells. The progeny of this animal harbor the abnormal DNA of the present invention in all of their germ cells and somatic cells. By preparing homozygous male and female animals having the introduced DNA in both homologous chromosomes and mating them, it can be insured that all their offsprings harbor the DNA.
The non-human mammal harboring the abnormal DNA of the present invention features a high expression of the abnormal DNA and, therefore, may eventually develop adiaphoria associated with functional inactivation of the protein of the present invention through inhibition of the function of the endogenous normal DNA and, therefore, can be utilized as an animal model of the disease. For example, by using the transgenic animal harboring the abnormal DNA of the present invention, analysis of the mechanism of this functional inactivation adiaphoria due to the protein of the present invention and therapeutic modalities for the disease can be explored.
As a specific potential use, the transgenic animal with a high expression of the abnormal DNA of the present invention can be used as a model for elucidating the functional inhibition of the normal protein by the abnormal protein of the present invention (dominant negative effect) in adiaphoria of functional inactivation type due to the protein of the present invention. Moreover, the transgenic mammal harboring the foreign abnormal DNA of the present invention develops symptoms due to an increase in the free protein of the present invention and, therefore, can be utilized in the screening of therapeutic compounds for functional inactivation of the protein of the present invention.
As other potential uses for transgenic animals harboring the two kinds of DNAs described above, the following uses can be suggested.
(1) Use as a cell source for tissue culture;
(2) Analysis of the relationship of the protein of the present invention to proteins which are specifically expressed or activated by the protein by direct analysis of DNAs or RNAs in the tissues of the transgenic mammal harboring the DNA of the present invention or analysis of the composition of the protein expressed by the DNA;
(3) Study of the functions of cells of those tissues which are generally difficult to culture by using the cells from the tissues containing the DNA as cultured by the standard tissue culture technique;
(4) Screening of drugs capable of enhancing the cell functions by using the cells described in (3);
(5) Isolation and purification of the muteins of the present invention and construction of antibodies to the muteins.
Furthermore, by using the transgenic animal of the present invention, clinical symptoms of diseases associated with the protein of the present invention, inclusive of said functional inactivation of the protein of the present invention, can be investigated. In addition, more detailed pathological findings can be generated in various organs of this model of diseases associated with the protein of the present invention, thus contributing to the development of new therapies and the study and treatment of secondary diseases arising from such diseases.
Moreover, following isolation of various organs from the transgenic animal of the present invention and their mincing and digestion with a proteolytic enzyme such as trypsin, free single cells harboring the transferred gene can be recovered and cultured for establishment of a cell line. Furthermore, characterization of cells producing the protein of the present invention can be made and their relationship to apotosis, differentiation, or proliferation, the mechanism of signal transduction in them, and abnormalities involved can be explored to thereby generate information useful for a further elucidation of the protein of the present invention and its actions.
Moreover, for the development of therapeutic drugs for diseases associated with the protein of the present invention, such as functional inactivation of the protein of the present invention by using the transgenic animal of the present invention, an effective and rapid screening technology for such therapeutic drugs can be established by using the test and assay methods described hereinbefore. In addition, by using the above transgenic animal or the foreign DNA expression vector of the present invention, gene therapies for diseases associated with the protein of the present invention can be explored and developed.
(8) Construction of Knockout Animals
The present invention further provides a non-human mammalian embryonic stem cell wherein the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention wherein the DNA is inactivated.
The present invention, therefore, provides:
(1) a non-human mammalian embryonic stem cell wherein the DNA of the present invention is inactivated;
(2) the non-human mammalian embryonic stem cell according to (1) wherein the DNA is inactivated by introduction of a reporter gene (e.g. a xcex2-galactosidase gene of the E. coli origin);
(3) the non-human mammalian embryonic stem cell according to (1) which is neomycin-resistant;
(4) the non-human mammalian embryonic stem cell according to (1) wherein the non-human mammal is a rodent;
(5) the non-human mammalian embryonic stem cell according to (4) wherein the rodent is a mouse;
(6) a non-human mammal deficient in expression of the DNA of the present invention, wherein the DNA is inactivated;
(7) the non-human mammal according to (6) wherein the DNA is inactivated by introduction of a reporter gene (e.g. a xcex2-galactosidase gene of E. coli origin) and the reporter gene can be expressed under the control of the promoter against the DNA of the present invention;
(8) the non-human mammal according to (6) wherein the non-human mammal is a rodent;
(9) the non-human mammal according to (8) wherein the rodent is a mouse; and
(10) a method for screening for a compound or a salt thereof which enhances or inhibits an activity of the promoter against the DNA of the present invention, which comprises administering a test compound to the non-human mammal according to (7) and detecting an expression of the reporter gene.
The term xe2x80x9cnon-human mammalian embryonic stem cell wherein the DNA of the present invention in inactivatedxe2x80x9d means the embryonic stem cell (hereinafter referred to briefly as ES cell) of a non-human mammal in which the DNA has been deprived of the capacity to express the protein of the present invention (hereinafter referred to sometimes as the knockout DNA of the present invention) through introduction of an artificial mutation to the DNA of the present invention possessed by the non-human mammal to thereby inhibit expression of the DNA of the present invention or through substantial deprivation of the activity of the protein of the present invention which is encoded by the DNA.
The non-human mammal includes the same animals mentioned hereinbefore.
Examples of the method for introducing an artificial mutation to the DNA of the present invention are a deletion of some or all of the DNA sequence, or an insertion or substitution of a different DNA by the genetic engineering technology. By such a mutation, the codon reading frame can be shifted or the function of the promoter or exon can be disrupted to provide the knockout DNA of the present invention.
The non-human mammalian embryonic stem cell wherein the DNA of the present invention is inactivated (hereinafter referred to as the ES cell wherein the DNA is the inactivated of the present invention or the knockout ES cell of the present invention) can be prepared by, for example, a procedure which comprises isolating the DNA of the present invention from an objective non-human mammal, inserting a drug-resistance gene, typically the neomycin-resistance gene or hygromycin-resistance gene, or a reporter gene such as lacZ (xcex2-galactosidase gene) or cat (chloramphenicol acetyltransferase gene) in its exon region to disrupt the function of the exon or inserting a DNA sequence for terminating gene transcription (e.g. poly A coupling signal) in the intron region between exons to thereby inhibit synthesis of a complete mRNA, introducing the thus-constructed DNA chain having a DNA sequence adapted to eventually disrupt the gene (hereinafter referred to briefly as the targeting vector) into the chromosomes of the host animal by homologous recombination, subjecting the resulting ES cell to Southern hybridization analysis using the DNA sequence on the DNA of the present invention or in its vicinity as the probe or a PCR procedure using the DNA sequence on the targeting vector and a DNA sequence in the vicinity but not including the DNA of the present invention used in the construction of the targeting vector as primers, and selecting the knockout ES cell of the present invention.
Moreover, the original ES cell used for inactivation of the DNA of the present invention by the homologous recombination technique or the like may be an already established cell line such as those mentioned hereinbefore or a new cell line established de novo by the known method of Evans and Kaufma. Taking murine ES cells as an example, ES cells of the 129 line are generally employed but the immunological background of this line is not clear. Therefore, the cell line established by using BDF1 mice created by the hybridization of C57BL/6 mice and C57BL/6 mice, both yielding few eggs, with DBA/2 mice (BDF1=F1 of C57BL/6 and DBA/2) for preparing pure-line ES cells with an immunologically defined genetic background can be used with advantage. In addition to the advantage of high egg output and sturdiness of the egg, BDF1 mice have the background of C57BL/6 mice so that in the construction of a disease model with ES cells obtained, the genetic background of the model mice can be converted to that of C57BL/6 mice by back-crossing with C57BL/6.
Moreover, in establishing an ES cell line, it is common practice to use blastocytes 3.5 days following fertilization but, aside from them, a large number of early embryos can be prepared with high efficiency by harvesting the embryos at the 8-cell stage and culturing them into blastocytes.
Furthermore, while ES cells from both male and female animals can be employed, generally ES cells of a male animal are more convenient for the construction of reproduction line chimeras. Moreover, for the purpose of reducing the burden of the complicated cultural procedure, it is preferable to carry out sexing as early as possible.
As a typical method for sexing ES cells, there can be mentioned the method in which the gene in the sex determination region on the Y chromosome is amplified and detected by PCR. Whereas the conventional karyotype analysis requires about 106 cells, the above method requires only about one colony equivalent of ES cells (about 50 cells). Therefore, the primary selection of ES cells in an early stage can be made by this sexing method. Since male cells can thus be selected in the early stage, the trouble in the initial stage of culture can be drastically reduced.
Moreover, the secondary selection can be carried out by G-banding for the number of chromosomes. The number of chromosomes in the resulting ES cell is preferably 100% of the normal number but this goal may not be reached due to the physical and other factors involved in the establishment of the line. In such cases, it is preferable to knockout the gene of the ES cell and reclone it in the normal cell (taking a mouse as an example, the cell in which the number of chromosomes is 2n=40).
The embryonic stem cell line thus established is generally very satisfactory in proliferation characteristic but since it is liable to lose its ontogenic ability, it must be subcultured with sufficient care. For example, this cell line should be cultured on suitable feeder cells such as STO fibroblasts in the presence of LIF (1-10000 U/ml) in a carbon dioxide incubator (preferably 5% CO2-95% air or 5% oxygen-5% CO2-90% air) at about 37xc2x0 C. and, in subculture, it should be treated with trypsin/EDTA solution. (generally 0.001-0.5% trypsin/0.1-5 mM EDTA, preferably about 0.1% trypsin/1 mM EDTA) to provide single cells and seed them on freshly prepared feeder cells. While such subculture is generally performed every 1-3 days, it is good practice to observe the cells on each occasion and, whenever morphologically abnormal cells are discovered, discard the culture.
ES cells can be allowed to differentiate into various types of cells, such as head long muscle cells, visceral muscle cells, heart muscle cells, etc. by conducting monolayer culture to a high density under suitable conditions or suspension culture until a mass of cells is formed (M. J. Evans and M. H. Kaufman, Nature, 292, 154, 1981; G. R. Martin, Proceedings of National Academy of Science USA, 78, 7634, 1981; T. C. Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985), and the cell deficient in expression of the DNA of the present invention as obtained by causing the ES cell of the present invention to differentiate is useful for the cytobiological in vitro study of the protein of the present invention.
The non-human mammal deficient in expression of the DNA of the present invention can be differentiated from the normal animal by assaying the mRNA in the animals by the known method and comparing the amounts of expression indirectly.
The non-human mammal used for this purpose includes the animals mentioned hereinbefore.
Referring to the non-human mammal deficient in expression of the DNA of the present invention, the DNA of the present invention can be knocked out by introducing the targeting vector constructed as above into, for example, almurine embryonic stem cell or a murine egg cell and thereby causing the DNA sequence of the targeting vector harboring the inactivated DNA of the present invention to undergo homologous recombination with, and accordingly replacing, the DNA of the present invention on the murine embryonic stem cell or egg cell chromosomes.
The cell with the DNA of the present invention thus knocked out can be obtained by Southern hybridization analysis using a DNA sequence on the DNA of the present invention or in its vicinity as a probe or by PCR using a DNA sequence on the targeting vector or a murine-derived DNA sequence in a region adjacent to but not including the DNA of the present invention used in the targeting vector as primers. When a non-human mammalian embryonic stem cell is used, a cell line with the DNA of the present invention knocked out by the homologous recombination technique is cloned and injected into the non-human mammalian embryo or blastocyte at a suitable stage of embryogenesis, for example at the 8-cell stage, and the resulting chimera embryo is transplanted in the pseudopregnant uterus of the non-human mammal. The animal thus obtained is a chimera animal constituted by both the cells harboring the normal DNA of the present invention and the cells harboring the artificially mutated DNA of the present invention.
When some of the gametes of this chimera animal harbor the mutated DNA of the present invention, an individual of which the entire tissues are constituted by cells harboring the mutated DNA of the present invention can be screened from the colony of animals obtained by crossing such a chimera animal with a normal animal, for example by coat color discrimination. The individuals thus selected are usually animals deficient in hetero-expression of the protein of the present invention and by mating such individuals deficient in hetero-expression of the protein of the present invention with each other, animals deficient in homo-expression of the protein of the present invention can be acquired.
When an egg cell is used, a transgenic non-human mammal with the targeting vector having been introduced into its chromosomes can be prepared by injecting the DNA solution into the egg cell nucleus by the microinjection technique and selecting animals expressing a mutation of the DNA of the present invention by homologous recombination.
The individuals with the DNA of the present invention knocked out are mated to verify that the animals obtained by mating also have the DNA knocked out and they can be sub-bred under the usual breeding conditions.
Preparation and maintenance of the reproduction line can also be carried out in the routine manner. Thus, by mating male and female animals harboring the inactivated DNA, homozygotes having the inactivated DNA in both homologous chromosomes can be obtained. The homozygotes thus obtained are bred under such conditions that, with regard to the dam, the number of homozygotes is plural per normal individual. By mating male and female heterozygotes, homozygotes and heterozygotes both harboring the inactivated DNA can be sub-bred.
The non-human mammalian embryonic stem cell harboring the inactivated DNA of the present invention is very useful for the construction of non-human mammals deficient in expression of the DNA of the present invention. Moreover, the mouse deficient in expression of the protein of the present invention lacks the various biological activities inducible by the protein of the present invention and can, therefore, be of use as an animal model of diseases arising from inactivation of the biological activities of the protein of the present invention, thus being of use in the etiological studies of diseases and development of therapeutics. (8a) A method for screening for a compound having an effect for treating or preventing in the various diseases caused by a defect in or damage to the DNA of the present invention
A non-human mammal deficient in expression of the DNA of the present invention can be used in the screening for a compound having an effect for treating or preventing in the diseases (e.g. arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity, hypertriglyceridemia, inflammatory diseases, senescence, diseases of brain, renal disorder, etc.) caused by a defect in or damage to the DNA of the present invention.
Thus, the present invention provides a method for screening for a compound, or a salt thereof, which has an effect for treating or preventing in the diseases caused by a defect in or damage to the DNA of the present invention, which method comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention and monitoring or measuring a change of the non-human mammal.
The non-human mammal deficient in expression of the DNA of the present invention, which is to be used in this screening method, includes the same animals as those mentioned above.
The test compound includes peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, blood plasma and so on. The test compound may be novel or known compounds.
More specifically, the non-human mammal deficient in expression of the DNA of the present invention is treated with the test compound and the treated animal is compared with an untreated control to evaluate the test compound for an effect for treating or preventing the diseases mentioned above by using a change in some organ or tissue or in a disease symptom as an indicator.
The method of treating the test animal with a test compound can be selected according to the symptom or symptoms manifested by the test animal and the characteristics of the test compound, among other factors and, for example, oral administration or intravenous injection can be employed. The dosage of the test compound can be suitably selected according to the route of administration, the properties of the test compound, and other conditions.
In the screening for a compound with an effect for treating or preventing arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity, hypertriglyceridemia, etc., for instance, the test compound is administered to the non-human mammals deficient in expression of the DNA of the present invention. And, changes in, blood glucose or body weight, etc. in the animal are determined at timed intervals.
When, in the screening method, blood glucose in the test animal is increased about 10% or more, preferably about 30% or more, more preferably about 50% or more, following administration of the test compound, the particular test compound can be selected as a compound capable of producing an effect for treating or preventing arteriosclerosis, atherosclerosis.
The compound obtained by the above screening method has an effect for treating or preventing the diseases (e.g. arteriosclerosis, atherosclerosis) caused by a defect in or damage to the protein, etc. of the present invention and, therefore, can be used as a drug, for example as a safe, low-toxicity agent for treating or preventing the diseases. Furthermore, compounds derived from the compound obtained by the above screening may also be used in the same manner.
The salts of the compound obtained by the screening method as mentioned above include salts with physiologically acceptable bases (e.g. alkali metals) or acids such as organic or inorganic acids, and are preferably a physiologically acceptable acid addition salt. Examples of such salts are salts thereof with inorganic acids (e.g. hydrochloric acid, phisphoric acid, hydrobromic acid or sulfuric acid, etc.) and salts thereof with organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid, etc.).
The composition for treating or preventing the diseases mentioned above comprising the compound obtained the screening method can be prepared in the same as the pharmaceutical composition comprising the protein, etc. of the present invention as mentioned above.
Because the thus-obtained preparation is safe and of low toxicity, it can be administered to humans or mammals (e.g., rat, mouse, guinea pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, etc.).
The dose of the compound may vary depending on subject disease, subject of administration, way of administration, and so on. When the compound is used, for example, for treating cancer by oral administration, the dose of the compound is normally about 0.1 to 100 mg, preferably 1.0 to 50 mg, and more preferably 1.0 to 20 mg per day for an adult human (weighing 60 kg). When the compound is used, for example, for treating arteriosclerosis, atherosclerosis by non-oral administration, it is advantageous to administer the compound in the form of injectable preparation at a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per administration by an intravenous injection for an adult human (weighing 60 kg), depending on subject of administration, subject disease and so on. For other animal species, corresponding doses as converted per 60 kg weight can be administered. (8b) A method for screening for a compound capable of promoting or inhibiting an activity of the promoter for the DNA of the present invention
The present invention provides a method for screening for a compound, or a salt thereof, which promotes or inhibits an activity of the promoter for the DNA of the present invention, which method comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention, wherein the DNA of the present invention is inactivated by introducing of a reporter gene and detecting the expression of the reporter gene.
As the test compound, the same compounds as those mentioned hereinbefore can be used.
Examples of the reporter gene are the same genes as those mentioned hereinbefore. Preferable examples are a xcex2-galactosidase gene (lacZ) and so on.
In non-human mammals deficient in expression of the DNA of the present invention wherein the DNA of the present invention is inactivated by introducing a reporter gene, the reporter gene is under the control of the promoter for the DNA of the present invention and, therefore, the activity of the promoter can be detected by tracing the expression of the substance encoded by the reporter gene.
For instance, when part of the DNA region coding for the protein of the present invention is inactivated by the Escherichia coli-derived xcex2-galactosidase gene (lacZ), xcex2-galactosidase is expressed in those tissues in which the protein of the present invention would have been expressed. Therefore, the status of expression of the protein of the present invention in a living animal body can be traced, easily and expediently, for example, by the staining method using a reagent serving as a substrate for xcex2-galactosidase, such as 5-bromo-4-chloro-3-indolyl-xcex2-galactopyranoside (X-gal). More specifically, a tissue section of a mouse defective in the protein of the present invention is fixed with glutaraldehyde or the like, washed with Dulbecco""s phosphate-buffered saline (PBS), and reacted with a staining solution containing X-gal at room temperature or around 37xc2x0 C. for about 30 minutes to 1 hour. The tissue sample is then washed with 1 mM EDTA/PBS solution to terminate the xcex2-galactosidase reaction and observed for color development. Alternatively, the mRNA coding for lacZ may be detected by a conventional method.
The compound, or a salt thereof, as obtained by the above screening method is a compound selected from among the test compounds mentioned above and, as such, is a compound capable of promoting or inhibiting the activity of the promoter for the DNA of the present invention.
The salts of the compound obtained by the screening method as mentioned above include salts with physiologically acceptable bases (e.g. alkali metals) or acids such as organic or inorganic acids, and are preferably a physiologically acceptable acid addition salt. Examples of such salts are salts thereof with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid, etc.) and salts thereof with organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid, etc.).
The compound, or a salt thereof, which promotes the activity of the promoter for the DNA of the present invention is capable of promoting the expression of the protein of the present invention and, hence, the promoting function of the protein. Therefore, the compound is useful as a drug, such as a safe, low-toxic agent for treating or preventing diseases such as arteriosclerosis, atherosclerosis, hyperlipidemia, hypercalorism, obesity, hypertriglyceridemia, inflammatory diseases, senescence, diseases of brain, renal disorder, etc.
On the other hand, the compound, or a salt thereof, which inhibits the activity of the promoter for the DNA of the present invention is capable of inhibiting expression of the protein of the present invention and, hence, inhibiting the function of the protein. Therefore, the compound is useful as a drug, such as a safe,.low-toxic agent for treating or preventing diseases such as malnutrition, abetalipoproteinemia or Tangier disease, etc.
Furthermore, compounds derived from the compound obtained by the above screening method may also be used in the same way.
The agent for treating or preventing the above-mentioned diseases comprising the compound obtained the screening method can be prepared in the same as the pharmaceutical composition comprising the protein, etc. of the present invention as mentioned above.
Because the thus-obtained preparation is safe and of low toxicity, it can be administered to humans or mammals (e.g., rat, mouse, guinia pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, etc.).
The dose of the compound may vary depending on subject disease, subject of administration, way of administration, and so on. When the compound which promotes an activity of the promoter is used, for example, for treating arteriosclerosis, atherosclerosis by oral administration, the dose of the compound which promotes an activity of the promoter is normally about 0.1 to 100 mg, preferably 1.0 to 50 mg, and more preferably 1.0 to 20 mg per day for an adult human (weighing 60 kg). When the compound which promotes an activity of the promoter is used, for example, for treating arteriosclerosis, atherosclerosis by non-oral administration, it is advantageous to administer the compound which promotes an activity of the promoter in the form of injectable preparation at a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per administration by an intravenous injection for an adult human (weighing 60 kg), depending on subject of administration, subject disease and so on. For other animal species, corresponding doses as converted per 60 kg weight can be administered.
When the compound which inhibits an activity of the promoter is used, for example, for treating abetalipoproteinemia by oral administration, the dose of the compound which inhibits an activity of the promoter is normally about 0.1 to 100 mg, preferably 1.0 to 50 mg, and more preferably 1.0 to 20 mg per day for an adult human (weighing 60 kg). When the compound which inhibits an activity of the promoter is used, for example, for treating abetalipoproteinemia by non-oral administration, it is advantageous to administer the compound which inhibits an activity of the promoter in the form of injectable preparation at a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per administration by an intravenous injection for an adult human (weighing 60 kg), depending on subject of administration, subject disease and so on. For other animal species, corresponding doses as converted per 60 kg weight can be administered.
In this way, the non-human mammal deficient in expression of the DNA of the present invention is very useful in view of the screening of a compound or a salt thereof which promotes or inhibits promoter activities of the DNA of the present invention, and useful for developing a pharmaceutical composition for treating or preventing various diseases arisen from deficiency in expression of the DNA of the present invention, and for analysis the in vivo mechanism of the various diseases.
Also, various kinds of DNAs coding proteins can be linked at downstream of the DNA comprising a promoter region of the LCAT-like protein of the present invention (e.g. the DNA comprising a nucleotide sequence represented by SEQ ID NO:38, substantially equivalent thereof, or a partial DNA thereof), to prepare a gene construct which can be microinjected into the fertilized egg of animals to produce transgenic animals. By using such transgenic animals, the mechanism of the protein in animals can be determined.
Moreover, by linking a suitable receptor gene at the above-mentioned promoter, and by constructing a cell-line which can produce the protein of the present invention, it can be used as an assay system to find low-molecular compounds which specifically promote or inhibit a producing activity of the LCAT-like protein in vivo.
Further, by analyzing the promoter mentioned above, it is possible to find a cis element or a transcription factor which can bind the promoter.
Examples of the DNA comprising substantially the same nucleotide sequence represented by SEQ ID NO:38, may be any DNA comprising the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:38 under a highstringent condition and having substantially the same promoter activities of the DNA having the nucleotide sequence represented by SEQ ID NO:38.
Examples of the DNA which comprises the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:38 under a highstringent condition are a DNA comprising a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:38.
The method of the hybridization and the highstringent condition are the same as mentioned above.
In the specification, claims and drawings of the present application, the abbreviations used for bases (nucleotides), amino acids and so forth are those recommended by the IUPAC-IUB Commission on Biochemical Nomenclature or those conventionally used in the art. Examples thereof are given below. Amino acids for which optical isomerism is possible are, unless otherwise specified, in the L form.
DNA: Deoxyribonucleic acid
cDNA: Complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: Cytosine
RNA: Ribonucleic acid
mRNA: Messenger ribonucleic acid
DATP: Deoxyadenosine triphosphate
dTTP: Deoxythymidine triphosphate
dGTP: Deoxyguanosine triphosphate
dCTP: Deoxycytidine triphosphate
ATP: Adenosine triphosphate
EDTA: Ethylenediaminetetracetic acid
SDS: Sodium dodecyl sulfate
Gly: Glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: Cysteine
Met: Methionine
Glu: Glutamic acid
Asp: Aspartic acid
Lys: Lysine
Arg: Arginine
His: Histidine
Phe: Phenylalanine
Tyr:Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
pGlu: Pyroglutamic acid
Substitution groups, protecting groups and reagents used in the specification of the present application are represented by the symbols set forth below.
Me: Methyl
Et: Ethyl
Bu: Butyl
Ph: Phenyl
TC: Thiazolidine-4(R)-carboxamide
Tos: p-Toluenesulfonyl
CHO: Formyl
Bzl: Benzyl
C12-Bzl: 2,6-Dichlorobenzyl
Bom: Benzyloxymethyl
Z: Benzyloxycarbonyl
Cl-Z: 2-Chlorobenzyloxycarbonyl
Br-Z: 2-Bromobenzyloxycarbonyl
Boc: tert-Butoxycarbonyl
DNP: Dinitrophenyl
Trt: Trityl
Bum: tert-Butoxymethyl
Fmoc: N-9-Fluorenylmethyloxycarbonyl
HOBt: 1-Hydroxybenzotriazole
HOOBt: 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide
DCC: Dicyclohexylcarbodiimide
SEQ ID NO:1 shows the amino acid sequence of a human protein of the present invention (matured), derived from human heart;
SEQ ID NO:2 shows the amino acid sequence of a human protein of the present invention (matured), derived from human kidney, wherein the 32 amino acids residues of 64th(Glu)-95th(Leu) in the amino acid sequence of SEQ ID NO:2 are inserted between the 63rd(Leu) and 64th(Val) in the amino acid sequence of SEQ ID NO:1;
SEQ ID NO:3 shows the amino acid sequence of a murine protein of the present invention (mature), derived from murine kidney;
SEQ ID NO:4 shows the amino acid sequence of a human protein of the present invention (mature), derived from human heart;
SEQ ID NO:5 shows the amino acid sequence of a human protein of the present invention (mature), derived from human kidney, wherein the 32 amino acids residues of 67th(Glu)-98th(Leu) in the amino acid sequence of SEQ ID NO:5 are inserted between the 66th(Leu) and 67th(Val) in the amino acid sequence of SEQ ID NO:4;
SEQ ID NO:6 shows the amino acid sequence of a human precursor protein of the present invention, derived from human heart;
SEQ ID NO:7 shows the amino acid sequence of a human precursor protein of the present invention, derived from human kidney, wherein the 32 amino acids residues of 97th(Glu)-128th(Leu) in the amino acid sequence of SEQ ID NO:7 are inserted between the 96th(Leu) and 97th(Val) in the amino acid sequence of SEQ ID NO:6;
SEQ ID NO:8 shows the amino acid sequence of a murine precursor protein of the present invention, derived from murine kidney;
SEQ ID NO:9 shows the amino acid sequence of a signal peptide of the present invention;
SEQ ID NO:10 shows the amino acid sequence of a signal peptide of the present invention;
SEQ ID NO:11 shows the amino acid sequence of a signal peptide of the present invention;
SEQ ID NO:12 shows the nucleotide sequence of a DNA coding for the human protein (mature) derived from a human heart having the amino acid sequence represented by SEQ ID NO:1 of the present invention;
SEQ ID NO:13 shows the nucleotide sequence of a DNA coding for the human protein (mature) derived from a human kidney having the amino acid sequence represented by SEQ ID NO:2 of the present invention;
SEQ ID NO:14 shows the nucleotide sequence of a DNA coding for the murine protein (mature) having the amino acid sequence represented by SEQ ID NO:3 of the present invention;
SEQ ID NO:15 shows the nucleotide sequence of a DNA coding for the human protein (mature) derived from a human heart having the amino acid sequence represented by SEQ ID NO:4 of the present invention;
SEQ ID NO:16 shows the nucleotide sequence of a DNA coding for the human protein (mature) derived from a human kidney having the amino acid sequence represented by SEQ ID NO:5 of the present invention;
SEQ ID NO:17 shows the nucleotide sequence of a DNA coding for the human protein derived from a human heart having the amino acid sequence represented by SEQ ID NO:6 of the present invention;
SEQ ID NO:18 shows the nucleotide sequence of a DNA coding for the human protein derived from a human kidney having the amino acid sequence represented by SEQ ID NO:7 of the present invention;
SEQ ID NO:19 shows the nucleotide sequence of a DNA coding for the human protein derived from a murine kidney having the amino acid sequence represented by SEQ ID NO:8 of the present invention;
SEQ ID NO:20 shows the nucleotide sequence of a DNA coding for the signal peptide having the amino acid sequence represented by SEQ ID NO:9 of the present invention;
SEQ ID NO:21 shows the nucleotide sequence of a DNA coding for the signal peptide having the amino acid sequence represented by SEQ ID NO:10 of the present invention;
SEQ ID NO:22 shows the nucleotide sequence of a DNA coding for the signal peptide having the amino acid sequence represented by SEQ ID NO:11 of the present invention;
SEQ ID NO:23 shows the partial nucleotide sequence of a DNA coding for the human protein of the present invention which is used for a cloning of the full length of DNA coding for the human protein of the present invention as shown in Example 1;
SEQ ID NO:24 shows the nucleotide sequence of a synthetic primer used for cloning of the DNA coding for the human protein of the present invention as shown in the Example 1;
SEQ ID NO:25 shows the nucleotide sequence of a synthetic primer used for cloning of the DNA coding for the human protein of the present invention as shown in the Example 1;
SEQ ID NO:26 shows the nucleotide sequence of a synthetic primer used for cloning of the DNA coding for the human protein of the present invention as shown in the Example 1;
SEQ ID NO:27 shows the nucleotide sequence of a synthetic primer used for cloning of the DNA coding for the murine protein of the present invention as shown in the Example 2;
SEQ ID NO:28 shows the nucleotide sequence of a synthetic primer used for cloning of the DNA coding for the murine protein of the present invention as shown in the Example 2;
SEQ ID NO:29 shows the partial nucleotide sequence of a DNA coding for the human protein of the present invention which is used for a cloning of the full length of DNA coding for the murine protein of the present invention as shown in Example 2;
SEQ ID NO:30 shows the nucleotide sequence of a synthetic primer used for cloning of the DNA coding for the murine protein of the present invention as shown in the Example 2;
SEQ ID NO:31 shows the nucleotide sequence of a synthetic primer used for cloning of the DNA coding for the murine protein of the present invention as shown in the Example 2;
SEQ ID NO:32 shows the nucleotide sequence of a synthetic primer used for cloning of the DNA coding for the murine protein of the present invention as shown in the Example 2;
SEQ ID NO:33 shows the nucleotide sequence of a synthetic primer used for cloning of the genomic DNA coding for the human protein of the present invention as shown in the Example 3;
SEQ ID NO:34 shows the nucleotide sequence of a synthetic primer used for cloning of the genomic DNA coding for the human protein of the present invention as shown in the Example 3;
SEQ ID NO:35 shows the nucleotide sequence of an adapter used for cloning of the genomic DNA coding for the human protein of the present invention as shown in the Example 3;
SEQ ID NO:36 shows the nucleotide sequence of a synthetic primer used for cloning of the genomic DNA coding for the human protein of the present invention as shown in the Example 3;
SEQ ID NO:37 shows the nucleotide sequence of a synthetic primer used for cloning of the genomic DNA coding for the human protein of the present invention as shown in the Example 3;
SEQ ID NO:38 shows the nucleotide sequence of 5xe2x80x2 flanking (a promoter sequence) of the genomic DNA coding for the human protein of the present invention;
SEQ ID NO:39 shows the amino acid sequence of the chemically synthesized partial peptide of human LCAT-like protein of the present invention as shown in the Example 5;
SEQ ID NO:40 shows the amino acid sequence of the chemically synthesized partial peptide of human LCAT-like protein of the present invention as shown in the Example 5;
SEQ ID NO:41 shows the nucleotide sequence of the primer for synthesizing the FLAG peptide as shown in the Example 7;
SEQ ID NO:42 shows the nucleotide sequence of the primer for synthesizing the FLAG peptide as shown in the Example 7;
SEQ ID NO:43 shows the amino acid sequence of the partial peptide as shown in the Example 11.
The transformant strain of Escherichia coli DH10B/pTB1972, which is obtained in the Example 1 mentioned hereinafter, is on deposit under the terms of the Budapest Treaty from Apr. 7, 1997, with the NIBH under the Accession Number of FERM BP-5900. It is also on deposit from Apr. 9, 1997 with the IFO under the Accession Number of IFO 16072.
The transformant strain of Escherichia coli DH10B/pTB1973, which is obtained in the Example 1 mentioned hereinafter, is on deposit under the terms of the Budapest Treaty form Apr. 7, 1997, with the NIBH under the Accession Number of FERM BP-5901. It is also on deposit from Apr. 9, 1997 with the IFO under the Accession Number of IFO 16073.
The transformant strain of Escherichia coli DH10B/pTB2010, which is in the Example 2 mentioned hereinafter, is on deposit under the terms of the Budapest Treaty from Jul. 8, 1997, with the NIBH under the Accession Number of FERM BP-6011. It is also on deposit from Jul. 9, 1997 with the IFO under the Accession Number of IFO 16111.
The transformant strain of Escherichia coli DH5xcex1/pTB2022, which is obtained in the Example 4 mentioned hereinafter, is on deposit under the terms of the Budapest Treaty from Jan. 20, 1998, with the NIBH under the Accession Number of FERM BP-6227. It is also on deposit from Jan. 19, 1998 with the IFO under the Accession Number of IFO 16154.