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|Zhu et al.
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|July, 2005
|Pea Line 08550848
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[0002] For example, bovine FSH is difficult to purify in substantial amounts from bovine pituitaries. Previous attempts to produce recombinant bovine FSH have resulted in a product that has been used in clinical trials, but it had little or no bioactivity. A relatively high yield of recombinant bovine FSH (rbFSH) can be obtained in insect cells by the baculovirus expression system, although sufficient upscaling of production has not been achieved yet. Application of (recombinant) gonadotrophin (such as FSH) in the human, for the purpose of assisted reproduction, has become common practice as is evident from the growing number of IVF clinics. Diagnostic methods to monitor treatment effects and to optimize protocols of injections, are routinely applied.
[0003] In the human field, TSH and recombinant versions thereof to stimulate thyroid tissue to overcome the need for elevating endogenous TSH after treatment against thyroid cancer. From a therapeutic perspective, there is considerable interest for the use of novel hTSH analogs. The creation of recombinant proteins as medicaments or pharmaceutical compositions by pharmaco-molecular agriculture constitutes one of the principal attractions of transgenic plants; it is also the domain where their utilization is accepted best by the public opinion. In addition to the yield and the favourable cost which may be expected from the field production of recombinant proteins for therapeutic purposes, transgenic plants present certain advantages over other production systems, such as bacteria, yeasts, and animal cells. Indeed, they are devoid of virus which might be dangerous to humans, and can accumulate the proteins of interest in their "organs of storage", such as seeds or tubers. This facilitates their handling, their transportation and their storage at ambient temperature, while affording the possibility of subsequent extraction according to needs.
[0004] Several heterologous proteins have successfully been produced in plants. Among these proteins are monoclonal antibodies, hormones, vaccine antigens, enzymes and blood proteins (Dieryck et al., 1997; Florack et al., 1995; Ma et al., 1995) Matsumoto et al., 1163; Saito et al., 1991; Thanavala et al. 1995) A major limitation of plants, shared with other heterologous expression systems like bacteria, yeast and insect cells, is their different glycosylation profile compared to mammals. In contrast to bacteria, having no N-linked glycans, and yeast, having only high mannose glycans, plants are able to produce proteins with complex N-linked glycans. Plant glycoproteins have complex N-linked glycans containing α1,3 linked core fucose and β1,2 linked residues not found in mammals (Lerouge et al., 1998) (
[0005] Schachter, 1991). A mouse monoclonal antibody produced in tobacco plants (Ma et al., 1995) has a typical plant N-glycosylation. 40% High-mannose glycans and 60% complex glycans containing xylose, fucose and 0, 1 or 2 terminal GlcNAc residues (Cabanes Macheteau et al., 1999).
[0006] In short, analyses of glycoproteins from plants have indicated that several steps in the glycosylation pathways of plants and mammals are very similar if not identical. There are however also clear differences, particularly in the synthesis of complex glycans. The complex glycans of plants are generally much smaller and contain beta-1,2 xylose or alpha-1,3 fucose residues attached to the Man3 (GlcNAc)2 core. Such residues on glycoprotein are known to be highly immunogenic. This will cause problems for certain applications of recombinant proteins carrying these sugars.
[0007] In addition, although common and often essential on mammalian glycoproteins, sialic acid has never been found in plant glycans. This is particularly relevant since experiments with both naturally FSH and recombinant FSH have shown, that the absence of terminal sialic acid on glycosidic side chains can decrease biological activity in vivo. Most likely, asialoglycoprotein-receptors in the liver can bind to asialo-FSH, and thereby cause a rapid clearance of the hormone from the circulation, which is reflected in a reduced metabolic half life and low bioactivity in vivo.
[0008] The invention provides a method to produce a glycoprotein hormone such as a thyroid-stimulating hormone or gonadotrophin or its corresponding receptor in a transgenic plant with modified glycosylation machinery, in order to allow for mammalian type of glycosidic side chains of the hormome such as the gonadotrophin and its corresponding receptor. In one embodiment of the invention, tobacco mosaic virus (TMV), the type member of the tobamovirus group of RNA viruses, is used as a viral vector for the expression of these recombinant hormones (in the detailed description gonadotrophins are mainly used) in these transgenic plants. In this expression system it has proven possible to achieve stable high level production of a number of heterologous proteins with desired glycosylation. Glycoprotein hormones, such as FSH, TSH, HCG, HMG, and PMSG, have essentially the alpha subunit in common, whereby the beta subunit effectively determines the specific activity of the hormone, and where here TSH and/or FSH are used, it its clear that also one of the other glycoprotein hormones is applicable.
[0009] In particular, the invention provides a method wherein stably transformed tobaccoplants with mammalian type of glycosylation are infected with modified TMV in order to produce bioactive rb TSH, rbFSH and rbFSH-R. For expressing recombinant bFSH both subunits of bFSH are inserted separately or together immediately downstream of an additional cp-promoter of TMV and subsequently checked for infectivity. For TSH, analogous methods are for example used, see below. In vitro transcripts are made and both constructs are rubbed mechanically onto the same plants susceptible for TMV: The resulting TMV particles, which can tolerate a larger than wild type genomic RNA can further spread throughout infected plants. Constructs are made which direct proteins into the secretory pathway.
[0010] For expression of bovine FSH receptor, a similar approach is used for preparation of the corresponding cDNA homologous oligonucleotide primers will be used to obtain overlapping cDNA fragments. Complete sequences for the receptor are reconstituted in a mammalian expression system. cDNA fragments encoding the N-terminal extracellular domains of the receptors are subcloned in the TMV vector in order to produce them as soluble receptors, fused to tag peptide for facilitation of their purification and further immobilization.
[0011] Another advantage in using TMV as a vector is the fact that the heterologous sequence is driven by a subgenomic promoter. The heterologous protein behaves completely independent from the virus, and can therefore be directed into different cellular compartments without interfering with the replication and expression of recombinant viral RNA.
[0012] In one embodiment of the invention, in order to modify plant glycosylation towards a more mammalian pattern, plant specific complex glycosylation is prevented by eliminating endogenous N-Acetylglucosaminyl transferase I (GnT I) activity. Downregulation/knocking out the GnT I gene is done by making transgenic plants that express the GnT I gene in sense or antisense orientation; these plants are analysed for their deficiency to add β1,2 xylose and α1,3 fucose.
[0013] Plants with no or little fucosyl or xylosyltransferase activity are used to express bFSH and its receptor using for example the TMV based expression vector.
[0014] Xylosyltransferase and fucosyltransferase can be knocked out and at least one of several mammalian glycosyltransferases have to be expressed. Providing the xylosyltransferase and fucosyltransferase knock-outs and thereby reducing the unwanted glycosylation potential of plants is a feasible option because for example an
[0015] In another embodiment, the invention provides a plant comprising a functional mammalian enzyme providing N-glycan biosynthesis that is normally not present in plants additionally comprising at least a second mammalian protein or functional fragment thereof that is normally not present in plants. It is provided by the invention to produce in plants a desired gonadotrophin or gonadotrophin-receptor having a mammalian-type of glycosylation pattern, at least in that said glycoprotein is galactosylated.
[0016] In a preferred embodiment, the invention provides a plant according to the invention wherein said functional mammalian enzyme providing N-glycan biosynthesis that is normally not present in plants comprises mammalian, such as human or bovine β1,4-galactosyltransferase. An important mammalian enzyme that is missing in plants is this β1,4-galactosyltransferase. cDNA's encoding this enzyme have been cloned from several mammalian species (Masri et al., 1988; Schaper et al., 1986). The enzyme transfers galactose from the activated sugar donor UDP-Gal in β1,4 linkage towards GlcNAc residues in N-linked and other glycans (
[0017] Surprisingly, the invention provides such a non-mammalian organism, in particular a plant having been provided with a functional mammalian enzyme providing N-glycan biosynthesis that is normally not present in plants, thereby for example providing the capacity to extend an N-linked glycan by the addition of a galactose. In this set of plants, formation of mammalian type of complex glycans is promoted by introducing mammalian glycosyltransferases. First, a 1,4-galactosyltransferase gene (β1,4 GT) is introduced in order to attach galactose to the Man3 (GlcNAc) 2 core. In a preferred embodiment, the α2,6-sialyltransferase (α2,6 ST) gene is introduced to the α1,4 GT plants to endow the N-glycans with terminal sialic acid residues. This is done by crossing transgenic α1,4-GT plants with transgenic α2,6 ST plants. The resulting plants function as hosts to produce recombinant gonadotrophin with terminal sialic acid and improved metabolic half life.
[0018] In a preferred embodiment, the invention provides such a plant wherein said enzyme shows stable expression. It is even provided that beyond said second mammalian protein a third mammalian protein is expressed by a plant as provided by the invention. The experimental part provides such a plant that comprises a nucleic acid encoding both an antibody light and heavy chain or fragment. Of course, it is not necessary that a full protein is expressed, the invention also provides a plant according to the invention expressing only a fragment, preferably a functional fragment of said second mammalian glycoprotein, such as a gonadotrophin or gonadotrophin-receptor, said fragment being characterised by for example a truncated polypeptide chain, or a not fully extended glycan, for example only extended with galactose.
[0019] In this invention functional fragments are understood to have at least one function in common with the original molecule. The activity should be of the same kind, not necessarily the same amount.
[0020] In a preferred embodiment, the invention provides a plant according to the invention wherein said second mammalian protein or functional fragment thereof comprises an extended N-linked glycan that is devoid of xylose and/or of fucose. As can be seen from for example
[0021] In the detailed description the invention provides a plant according to the invention, in particular a tobacco plant, or at least a plant related to the genus Nicotiana. However, use for the invention of other relatively easy transformable plants, such as
[0022] Herewith, the invention provides a method for providing a transgenic plant, such as transgenic Nicotiana,
[0023] With said method as provided by the invention, the invention also provides a plant expressing said recombinant protein and expressing a functional mammalian enzyme involved in mammalian N-glycan biosynthesis that is normally not present in plants. Now that such a plant is provided, the invention also provides use of a transgenic plant to produce a desired glycoprotein or functional fragment thereof, such as gonadotrophin or gonadotrophin-receptor, in particular wherein said glycoprotein or functional fragment thereof comprises an extended N-linked glycan et least comprising galactose.
[0024] The invention additionally provides a method for obtaining a desired gonadotrophin or gonadotrophin-receptor or functional fragment thereof comprising for example an extended N-linked glycan at least comprising galactose; comprising cultivating a plant according to the invention until said plant has reached a harvestable stage, for example when sufficient biomass has grown to allow profitable harvesting, followed by harvesting said plant with established techniques known in the art and fractionating said plant with established techniques known in the art to obtain fractionated plant material and at least partly isolating said glycoprotein from said fractionated plant material. In the detailed description (see for example
[0025] The invention thus provides a plant-derived gonadotrophin or gonadotrophin-receptor or functional fragment thereof comprising an extended N-linked glycan at least comprising galactose, for example obtained by a method as explained above. The invention furthermore provides a plant-derived and at least partly purified gonadotrophin, such as bovine FSH, and its corresponding receptor, from transgenic plants infected with a recombinant virus such as modified TMV. Immuno-affinity chromatography combined with Centricon filtration has been shown to be an effective purification method in the case of insect cell-derived recombinant bFSH. Similar methods are developed for plant-derived rbFSH. Gonadotrophin-receptor is purified either by affinity chromatography (with insolubilized ligand or specific antisera) or by electro-elution after polyacrylamide gel electrophoresis. If productions are very abundant, classical separation techniques (size, hydrophobicity, etc.) can be used. Homologous transfected cell lines expressing receptors for bFSH are developed for in vitro measurement of bioactive recombinant bovine FSH. Several other types of assays are available already for monitoring of rbFSH during production and purification. For monitoring of gonadotrophin-receptor production and purification, assays have already been developed [immuno radio metric assay (IRMA), Western blotting] with respect to receptors of the porcine species. Likewise, these assays can be used for the measurement of bovine receptors. Herewith, the invention also provides use of such a plant-derived gonadotrophin or gonadotrophin-receptor or functional fragment thereof according to the invention for the production of a pharmaceutical composition, for example for the treatment of a patient with an reproductive disorder. Such a pharmaceutical composition comprising a plant-derived gonadotrophin or gonadotrophin-receptor or functional fragment thereof is now also provided. The invention furthermore provides plant-derived recombinant gonadotrophin such as bFSH and its receptor as very pure, stable and specific reagents in an assay method of commercial significance and as a therapeutic tool for assisted reproduction. The availability of plant-derived bovine gonadotrophins (FSH and LH), and of diagnostic testkits for the measurement of these substances provides a means of overcoming the limitations on assisted reproduction in for example cattle, as imposed by impure agents and lack of diagnostic tools. In animal reproduction, FSH is employed for increased production of eggs in cattle and for treatment of infertility in cattle and pigs.
[0026] In addition, the invention provides a plant comprising a cell comprising a functional mammalian enzyme or functional fragment thereof providing N-glycan biosynthesis additionally having been provided with an expression vector comprising a nucleic acid encoding a thyroid-stimulating hormone (TSH) or functional fragment thereof. TSH is another member (like FSH) of the glycoprotein hormone family (Grossmann et al, 1997, Endocrine Review, p 476-500) and essentially has the alpha subunit in common with FSH, which makes the FSH methods and plants provided herein of course easily applicable to the TSH field, if desired in combination with skills available in the art in said field.
[0027] Such a rbTSH plant as provided herein is preferably equipped with the enzyme human α1,4-galactosyltransferase, allowing the production of thyroid-stimulating hormone or functional fragment thereof that comprises an extended N-linked glycan, preferably galactose.
[0028] In another embodiment as provided herein, such rbTSH plant is essentially devoid of xylose and or fucose. Such a plant is obtained quit along the lines for rbFSH plants, whereby said expression vector is derived from a plant virus,for example a tobamovirus such as tobacco mosaic virus, which is most easily used with a tobacco plant. Of course, use of a rbTSH plant to produce a desired thyroid-stimulating hormone or functional fragment thereof, which preferably comprises an extended N-linked glycan et least comprising galactose, is also provided.
[0029] Furthermore, the invention provides a method for obtaining a thyroid-stimulating hormone or functional fragment thereof comprising cultivating a rbTSH plant as provided herein until said plant has reached a harvestable stage, harvesting and fractionating said plant to obtain fractionated plant material and at least partly isolating said thyroid-stimulating hormone or fragment thereof from said fractionated plant material. A plant-derived thyroid-stimulating hormone or functional fragment thereof comprising an extended N-linked glycan at least comprising galactose is hereby thus provided Furthermore, the invention provides use of a recombinant thyroid-stimulating hormone or functional fragment thereof according to the invention for the production of a pharmaceutical composition. In particular such use according to the invention is provided for the production of a pharmaceutical composition for the treatment of a thyroid dysfunction and a pharmaceutical composition comprising a thyroid-stimulating hormone or functional fragment thereof according to the invention
[0030] The invention is further explained in the detailed description without limiting it thereto.
[0031] One important enzyme involved in mammalian N-glycan biosynthesis that is not present in plants is β1,4-galactosyltransferase. Here, for one, the stable expression of β1,4-galactosyltransferase in tobacco plants is described. The physiology of these plants is not obviously changed by introducing α1,4-galactosyltransferase and the feature is inheritable. Crossings of a tobacco plant expressing β1,4-galactosyltransferase with a plant expressing the heavy and light chain of a mouse antibody produced antibody having terminal galactose in similar amounts as hybridoma produced antibodies. Herein it is thus shown that the foreign enzyme can be successfully introduced in plants. A clear increase in galactose containing glycoproteins is observed. Moreover, this feature is inheritable and there is no visible phenotypical difference between the galactosyltransferase plants and wild type. A mouse monoclonal antibody produced in these plants has a degree of terminal galactoses comparable to hybridoma produced antibody. This shows that not only endogenous proteins become galactosylated but also a recombinantly expressed mammalian protein.
[0032] Materials and Methods
[0033] Plasmids and Plant Transformation
[0034] A plant transformation vector containing human β1,4-galactosyltransferase was constructed as follows: a 1.4 kb BamHI/XbaI fragment of pcDNAI-GalT (Aoki et al., 1992; Yamaguchi and Fukuda, 1995) was ligated in the corresponding sites of pUC19. Subsequently, this fragment was re-isolated using surrounding KpnI and HincII sites and cloned into the KpnI and SmaI site of pRAP33 (named pRAP33-HgalT). Using AscI and PacI sites the CaMV35S promotor-cDNA-Nos terminator cassette of pRAP33-HgalT was cloned in the binary vector pBINPLUS (van Engelen et al., 1995). Modifications to the published protocol are: After incubation with
[0035] Northern Blotting
[0036] The β1,4-galactosyltransferase RNA level in the transgenid plants was analyzed by northern blotting (Sambrook et al., 1989) RNA was isolated from leafs of transgenic and control plants as described (De Vries et al., 1991). Ten μg of total RNA was used per sample. The blot was probed with a [
[0037] Glycoprotein Analysis
[0038] Total protein extracts of tobacco were prepared by grinding leafs in liquid nitrogen. Ground material was diluted 10 times in SDS page loading buffer (20 mM of This-HCl pH 6.8, 6% glycerol, 0.4% SDS, 20 mM DTT, 2.5 ìg/ml Bromophenol Blue). After incubation at 100° C. for 5 min insoluble material was pelleted. Supernatants (12.5 μl/sample) were run on 10% SDS-PAGE and blotted to nitrocellulose. Blots were blocked overnight in 0.5% Tween-20 in TBS and incubated for 2 hours with peroxidase conjugated RCA
[0039] Plant Crossings
[0040] Mgr48 (Smant et al., 1997) is a mouse monoclonal IgG that has been expressed in Tobacco plants. The construct used for transformation was identical to monoclonal antibody 21C5 expressed in tobacco (van Engelen et al., 1994). Flowers of selected tobacco plants with high expression of β1,4-galactosyltransferase were pollinated with plants expressing Mgr48 antibody. The F1 generation was seeded and plants were screened for leaf expression of antibody by western blots probed HRP-conjugated sheep-anti-mouse and for galactosyltransferase expression by RCA as described above.
[0041] Purification of IgG1 from Tobacco
[0042] Freshly harvested tobacco leaves were ground in liquid nitrogen. To 50 g of powdered plant material, 250 ml of PBS, containing 10 mM Na
[0043] Purified antibody was quantified by comparison of the binding of HRP-conjugated sheep-anti-mouse to the heavy chain on a western blot with Mgr48 of known concentration purified from hybridoma medium (Smant et al., 1997).
[0044] Hybridoma Mgr48 and plant produced Mgr48 was run on 10% SDS-PAGE and blotted as described above. Detection with RCA was as described above. For antibody detection, blots were probed with HRP-conjugated sheep-anti-mouse and detected with Lumi-Light western blotting substrate as described above.
[0045] Results
[0046] Human β1,4-galactosyltransferase galactosylates Endogenous Proteins in Nicotiana Tobacum.
[0047] Human α1,4-galactosyltransferase (Masri et al., 1988) was introduced in tobacco plants by Agrobacterium mediated leaf disk transformation of plasmid pBINPLUS-HgalT containing a cDNA that includes a complete coding sequence. Twenty-five plants selected for kanamicin resistance were analysed for mRNA levels by northern hybridization (
[0048] As it is known that galactosylated N-glycans are poor acceptors for plant xylosyl- and fucosyltransferase (Johnson and Chrispeels, 1987), the influence of expression of β1,4-galactosyltransferase on the occurrence of the xylose and fucose epitope was investigated by specific antibodies. A polyclonal rabbit anti-HRP antibody that reacts with both the xylose and fucose epitope shows a clear difference in binding to isolated protein from both control and transgenic plants (
[0049] Recombinantly Produced Antibody is Efficiently Galactosylated.
[0050] The effect of expression of β1,4-galactosyltransferase on a recombinantly expressed protein was investigated. Three tobacco plants expressing β1,4-galactosyltransferase (no. GalT6, GalT8 and GalT15 from
[0051] Several were found in crossing GalT8xmgr48. Two of these plants (no.
[0052] Using proteinG affinity, antibody was isolated from tobacco plants expressing mgr48 and from the two selected plants expressing both mgr48 and β1,4-galactosyltransferase. Equal amounts of isolated antibody was run on a protein gel and blotted. The binding of sheep-anti-mouse-IgG and RCA to mgr48 from hybridoma cells, tobacco and crossings GalT8xmgr48-11 and 12 was compared (
[0053] There is a need for an accessible and standardised source of FSH for therapeutic and diagnostic purposes, which is guaranteed to be free of LH activity.
[0054] FSH preparations normally are derived from ovine or porcine pituitaries, which always implies the presence of (traces of) LH, and the risk of contamination with prion-like proteins. Substitution of brain derived FSH for plant produced recombinant FSH may be a good method of eliminating these problems.
[0055] Furthermore application of plant produced FSH receptor (FSHR) in a diagnostic testkit provides a good method for measurement of bioactive FSH by receptor assay. However, production of bioactive animal glycoproteins in plants, especially for therapeutic purposes, requires modification of plant-specific sugar sidechains into a mammalian type of glycans. The invention provides recombinant bFSH and bFSHR by infecting stably transformed tobaccoplants capable of forming mammalian type of glycans, with recombinant Tobacco Mosaic Virus TMV containing the genes for bFSH or bFSHR.
[0056] Construction of Single Chain (sc) bFSH into pKS (+) Bluescript Vector, Construction of sc-bFSH-TMV and sc-bFSH-HIS-TMV In order to circumvent the need of simultaneous expression of the two separate genes of bFSH-alpha and bFSH-beta subunits in plants, we decided to construct a bFSH fusion gene.
[0057] By overlap PCR we fused the carboxyl end of the beta subunit to the amino end of the alpha subunit (without a linker). In addition, we constructed a second sc-bFSH version carrying a 6× HIS tag at the C-terminus of the alpha subunit, which will allow us to purify the recombinant protein from the plant. Both, sc-bFSH and sc-bFSH-HIS constructs were subcloned into the cloning vector pKS(+) bluescript. The correctness of the clones was confirmed by sequence analysis.
[0058] Sc-bFSH was subcloned into the TMV vector. Two positive clones were chosen to make in vitro transcripts and Inoculate
[0059] Molecular Cloning of Full Length cDNA Encoding the Bovine FSH Receptor; Cloning of the Extracellular Domain of the FSH Receptor in TMV Vector.
[0060] Oligonucleotide primers based on partial published sequence date were designed for PCR amplification of nucleotide 1 to 1100 and 650 to 2150, respectively, from a bovine testicular cDNa library. The two fragments were subcloned in the pGEM-T vector (Stratagene), and fully sequenced. A unique common internal restriction site (Xbal) allowed the fragment ligation while subcloning into the eukaryotic expression vector pEE14. After plasmid amplification of a recombinant clone, transfection of CHO-K1 cells is the next step. Stable transfectants are usually obtained in three weeks. Functional experiments (hormone specific binding and transduction) will allow selection of the best expressing clones before the amplification process with increasing amounts of MSX in cell media.
[0061] In order to obtain a soluble FSH receptor, a fragment encoding part of its N-terminal extracellular domain was obtained by using PCR. The size of the soluble receptor (293 aminoacids) has been chosen in order to retain all hormone interaction, and favor processing by elimination of the C-terminal Cystein cluster. Amplimers bearing appropriate restriction sites for subcloning the TMV vector were designed. After amplification and cloning, a synthetic DNA encoding the FLAG epitope (sequence=DYKDDDDK) as well as a stop codon was ligated. Subcloning of the construct into the TMV vector is now in progress.
[0062] In order to express the bovine FSH in a transient plant expression system the following constructs were cloned in a TMV expression vector:
[0063] the individual subunits of bFSH: α and β
[0064] a bFSH single chain (sc-bFSH) with the carboxy end of the β subunit fused to the amino end of the α subunits (according to Sugahara et al., 1996)
[0065] the single chain tagged with 6×HIS at the carboxy end (sc-bFSh-HIS)
[0066] After inoculation of
[0067] By reverse transcription-PCR analysis we could demonstrate the in planta stability of the hybrid TMV genomes carrying the bFSH sequences. Surprisingly also the co-transfected alpha and beta bFSH constructs were stably propagated in the same plants.
[0068] For detection of recombinant bFSH Western blot analysis of crude protein extracts from leaf material was carried out. Using an anti-human FSH beta subunit antiserum (
[0069] The presence of the 6×HIS tag on the sc-bFSH could be demonstrated using anti-HIS monoclonal antibodies. In a small scale protein miniprep using Ni-NTA agarose we were able to purify the sc-bFSH-HIS under denaturing conditions.
[0070] In order to investigate whether the sc-bFSH is glycosylated or not an enzymatic glycan digestion, PNGaseF digestion, was carried out. A clear band shift of bFSH treated with PNGaseF on Western blot analysis indicated the presence of N-glycans.
[0071] As sc-bFSH was detected almost exclusively in the soluble fraction after fractionating crude protein extracts with 100.000×g, clearly the sc-bFSH is secreted by, the plant cells into the extracellular space. Intercellular washing fluit (IF) extractions from leaf material were carried out. As shown by Western blot analysiss the sc-bFSH was clearly enriched in these IF fractions indicating a secretion of protein into the extracellular space.
[0072] Expression of Biologically Active Glycoforms of Bovine Follicle Stimulating Hormone in Plants.
[0073] The follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone which regulates the ovarian follicle and testicular tubule development in all vertebrate species. In particular ovine, porcine or equine FSH are widely used to induce superovulation for human assisted reproduction of cattle and can benefit from homologous (i.e. bovine) recombinant FSH being free of potentially infectious material and other contaminating hormones. Here we describe the application of a plant based transient expression system for the rapid production of bFSH and its biochemical, immunological and biological. characterisation. We have used a tobacco mosaic virus-based vector to express bFSH in the tobacco related species
[0074] The follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone which regulates the ovarian follicle and testicular tubule development in all vertebrate species (Pierce and Parsons, 1981; Bielinska and Boime, 1995).
[0075] Together with luteinizing hormone (LH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG), FSH forms the glycoprotein hormone family, which is the structurally most complex hormone family in the animal kingdom.
[0076] These hormones are composed of two non covalently associated subunits, a common alpha subunit, and an unique beta subunit which confers biological specificity to each of these hormones. Each subunit forms intrachain disulfide bridges and carries two N-linked oligosaccharides, which is necessary for proper folding and secretion of the hormones (Suganuma et al., 1989, Feng et al., 1995). The N-linked carbohydrate chains of FSH exhibit considerable variation in both size and structure, including the degree of terminal sialyation and/or sulfation (Baenzinger and Green, 1988). The functional significance of this diversity of isoforms is not yet fully understood, but sialic acid seems to be the major determinant for the circulatory stability of FSH by preventing its rapid clearance mediated by the hepatic asialo-glycoprotein receptor (Drickamer, 1991). Besides this influence upon plasma halflife, the glycan heterogeneity is thought to provide a fine tuning mechanism with which to control gonadal function.
[0077] The technique of superovulation and embryo transfer is widely used to increase the number of offspring of genetically superior cows. Induction of superovulation is usually performed using pregnant mare serum gonadotropin (PMSG) or pituitary derived FSH. Apart from containing potentially infectious viral or prion material, these preparations have the disadvantage of always containing some LH which is thought to contribute to the high variation in the results of treatments (Wilson J M et al., 1993).
[0078] To overcome these problems, an attempt was made to produce recombinant bovine FSH (rbFSH) in the baculovirus expression system (van de Wiel et al., 1998). Two main problems were associated with rbFSH: first, the glycans apparently did not contain terminal sialic acid, due to a probable complete inability of the insect cell line to perform sialyation (for a review see: Altmann et al., 1999). Second, although a relatively high yield of rbFSH was obtained in the baculovirus expression system, sufficient upscaling of production has not been achieved yet, especially because a decrease of expression rates in large scale fermentations using higher cell densities (Taticek et al., 1994).
[0079] In the recent years, the expression of recombinant proteins in plants has become a matter of interest. As an eucaryotic system, plant cells are capable of targeting recombinant proteins to the secretory pathway and of carrying out posttranslational modifications including disulfide bridge formation and glycosylation (Hiatt et al. 1989; Ma et al., 1995, Cabanes-Macheteau 1999). The N-glycosylation in higher organisms is conserved but differs in details. The processing of N-linked glycans occurs along the secretory pathway and complex-type N-glycans arise and are modified in the Golgi apparatus. Since some of the modifications are specific for an expression system, the structure of mature complex N-glycans differ to some extent in plants and mammals. In particular, plant glycoproteins do not bear sialic acid and carry a(1,3)-fucose and b(1,2)-xylose attached to their proximal N-acetylglucosamine which have not been found in mammals (for recent review see Lerouge et al., 1998). Since plants are gaining acceptance for the expression of recombinant therapeutic proteins (e.g. Ma et al., 1998 Tacket et al., 2000), it is important to examine in detail to what extent glycans of mammalian glycoproteins produced in plants differ from the original ones, and could influence their physiological properties. In this instance, glycoprotein hormones offer a particularly demanding model since proper N-glycosylation is required for folding, subunit assembly, intracellular trafficing and biological activity.
[0080] In this study, we used a plant viral vector to transiently express the bovine FSH in the tobacco related species
[0081] Material and Methods
[0082] Construction of p4GD-sc-bFSH
[0083] In order to construct the single chain bFSH (sc-bFSH) with the carboxyl end of the b-subunit fused to the amino end of the a subunit (Sugahara et al., 1996) a gene SOEing strategy (Horton, 1993) was chosen (
[0084] Plant Material and Inoculation of Plants
[0086] Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis of Infected Plants
[0087] 12-16 dpi the replicative stability of the hybrid TMV RNA—genome derived from p4GD-sc-bFSH was investigated. Total RNA from systemically infected leaves was prepared using TriReagent (Molecular Research Centre, Inc.) and cDNA synthesis (reverse transcription) was performed using the TMV (p4GD-PL) specific reverse primer p4GD-RV 5′-TTT TTC CCT TTT TTG TTT TCC G-3′ located downstream the multiple cloning site. Using p4GD-RV and the TMV specific forward primer p4GD-FW 5′-GAT GAT GAT TCG GAG GCT ACT-3′ which anneals upstream of the multiple cloning site, a specific RT-PCR fragment of 826 bp was expected for the sc-bFSH construct. As negative control the same procedure was also carried out on wt TMV (p4GD-PL) infected plants. In this case a RT-PCR product of 145 bp was expected.
[0088] Total Soluble Protein Extraction from sc-bFSH Expressing
[0089] Two to three weeks after inoculation, systemically infected leaves were harvested, and total soluble protein (TO) was extracted by grinding the leaves in 10 vols (w/v) of 10 mM TRIS-HCl pH 7.6 buffer. Cellular debris were sedimented (15 min, 6000 rpm, 4° C.). The supernatant was used for SDS-PAGE. The same procedure was also carried out on leaves which were subjected to intercellular washing fluid extraction (see below).
[0090] Preparation of Intercellular Washing Fluid (IF) from sc-bFSH Expressing Leaves
[0091] In order to enrich periplasmic (extracellular) proteins an intercellular washing fluid (IF) extraction was carried out using the method described by Pogue et al. (1997) with modifications. Systemically infected leaves from sc-bFSH expressing plants, were harvested two to three weeks after inoculation. The tissue was rolled lengthwise in parafilm® (American National Can, IL, USA), inserted into a 50 ml plastic tube and submerged in pre-cooled (4° C.) 10 mM TRIS-HCl pH 7.6 buffer. A vacuum was applied twice for 1 min, with a rapid release of the vacuum to infiltrate the leaves with buffer. Subsequently, the leaves were blotted dry to remove excess buffer, were again rolled in parafilm and inserted into a centrifuge tube. The IF was collected by a downspin at 3,000×g for 15 min at 4° C. The IF was finally clarified by a centrifugation at 10.000×g for 15 min at 4° C. and the supernatant used for analyses. The same IF preparations were made from plants infected with p4GD-PL (wt TMV) or not infected wildtype plants serving as negative controls.
[0092] Fractionation of sc-bFSH Containing Plant Extracts by Ultracentrifugation
[0093] Crude total protein extracts (TO) from sc-bFSH expressing plants were prepared as described above, followed by ultracentrifugation at 100.000×g for 1 h at 4° C. to obtain a pellet (P) and a supernatant (SN) fraction. The pellet was resuspended in 10 mM TRIS-HCl pH 7.6 in the same volume as the supernatant fraction. Equal volumes of total extract, pellet and supernatant fractions were subjected to Western blot analysis (see below).
[0094] N-glycosidase F Digestion of sc-bFSH
[0095] The procedure was as described by Tretter et al., 1991 with modifications. IF extraction from sc-bFSH expressing plants (see above) was conducted using 50 mM TRIS-HCl, 20 mM EDTA, pH 8.0, followed by an addition of b-mercaptoethanol and SDS to each 0,5% (v/v, w/v, respectively). 10 ml of this extract were heated at 95° C. for 5 min, followed by an addition of 40 ml 50 mM TRIS-HCl, 20 mM EDTA, pH 8.0 and 1% IGEPAL (Sigma, MI, USA). Finally, 1 ml of N-glycosidase F, peptide-N4-[N-acetyl-b-glucosaminyl] asparagine amidase, (20 mU/ml; Roche, CH) was added, followed by an incubation at 37° C. overnight (16 h). After digestion, the proteins were precipitated using aceton and subjected to Western blot analysis. As a control, the same procedure was done without the addition of N-glycosidase F.
[0096] Western Blot Analysis of sc-bFSH Extracts
[0097] Protein extracts prepared as described above were electrophoresed on 12.5% SDS-polyacrylamide gels under reducing conditions (Lämmli, 1970). Following electroblotting onto nitrocellulose (Amersham Life Science Ltd, U.K.), the blots were blocked with 5% non-fat dry milk in TBS containing 0,1% Tween 20 (Sigma, MI, USA). The primary antibody was an anti-human FSH b-subunit (R812, reference!) rabbit polyclonal antiserum diluted to 1:2500 in TBS containing 0,1% Tween (TTBS) and 1% BSA (Sigma, MI, USA). As secondary antibody an anti-rabbit IgG goat polyclonal antiserum-horde radish peroxidase conjugate (Sigma, MI, USA), diluted to 1:20000 in TTBS, was employed. Detection was done using an enhanced chemiluminescence substrate (ECL, Super Signal, Pierce, Ill., USA). For quantification of the signal intensities Kodak Digital Science 1D Image Analysis Software was used.
[0098] In parallel to the immunodetections, the total protein contents of the different extracts were visualised using silver staining of the gels (Amersham Pharmacia Biotech AB). Quantitation of total protein was done using a BCA protein assay kit (Pierce, Ill., USA).
[0099] Localisation of sc-bFSH by in situ Indirect Immunofluorescence
[0100] Indirect in situ immunofluorescence was performed according to Goodbody et al. (1994) and Flanders et al. (1990) with modifications. All solutions were made in microtubule stabilizing buffer (MTSB): 50 mM PIPES, 5 mM EGTA, and 5 mM MgSO
[0101] Microscopic Imaging
[0102] Imaging was conducted on a Biorad MRC 600 confocal laser scanning microscope equipped with a Krypton/Argon mixed gas laser and a ×40 objective. Excitation of the Cy3 fluorochrome was done at 569 nm using the YHS filter block. Phase contrast illumination of the tissue sections was performed on the same sections. Images from the confocal system were imported into PaintShopPro 5.01 (Jasc Software, Inc., MN, USA) for colorisation.
[0103] Immunoaffinity Chromatography
[0104] Immunoaffinity chromatography was essentially done as described previously (Van de Wiel et al., 1998) and according to the mannufacturer's instructions. The gel pellet was washed 2 times with PBS13 and then incubated with 30 ml of sc-bFSH The purity and concentration of sc-bFSH in the eluate fractions were monitored on a silver stained SDS-PAGE-gel, on which the sc-bFSH appeared as a single band. For mass spectrometry analysis (see below), a volume of pure sc-bFSH corresponding to approximately 8 mg was first dialyzed against deionized water using Slide-a-lyzer® MINI dialysis units having a molecular weight cut off of 10 kDa (Pierce, Ill., USA) to remove salts and glycine. Subsequently, the dialyzed fractions were concentrated by lyophilisation, and dissolved in SDS-PAGE sample buffer.
[0105] N-glycan Analysis of sc-bFSH by Matrix Assisted Laser Desorption/ionisation Mass Spectrometry (MALDI-MS)
[0106] The procedure was carried out as described by Kolarich and Altmann (in press) with modifications. In brief, approximately 8 mg of immunoaffinity—purified sc-bFSH were electrophoresed on a reducing 12.5% C 1%T SDS-polyacrylamide gel system. Following electrophoresis, the gel was stained using a silver staining method which is compatible with mass spectrometry analysis (Schevchenko et al. 1996). The band of interest was excised with a scalpel, and after washing, reduction and S-carboxamidomethylation subjected to tryptic in-gel digestion as described by Jensen et al. (1997). In order to identify the sc-bFSH, the extracted peptides were dissolved in 10 ml 5% (v/v) formic acid and analysed on a DYNAMO (ThermoBioAnalysis, Ltd.) linear time-of-flight MALDI-MS (peptide mapping). 0.2 ml of the sample was dried on the plates followed by addition of 0.8 ml matrix solution (1% (w/v) a-cyano-4-hydroxycinnamic acid in 70% (v/v) acetonitrile. Peptides were measured with a <<dynamic extraction>> setting 0.1. Average masses of [M+H]
[0107] Following peptide mapping, the N-glycans in the residual aliquot of the tryptic digest were released by peptide-N
[0108] In vitro Bioactivity Assay in FSH Receptor Expressing CHO Cells
[0109] IF-extracts from
[0110] Superovulatory Treatment of Mice
[0111] 15 6-8 week old female C57/CBA mice were treated each with 100 ml of 11 times concentrated sc-bFSH-IF extract (concentration was done using an AMICON ultrafiltration cell, MWCO 10 kDa). Serving as a negative control, 15 mice were treated each with 100 ml of 11 times concentrated IF-extract from not infected plants (NI-IF). Furthermore, the response of 14 mice to 5IU pregnant mare serum gonadotropin (PMSG, Folligon, Intervet) in the same volume was investigated. 46-48 hours post FSH or wt-IF injections, the 3 groups were treated with 5 IU human chorionic gonadotropin (hCG ; Chorulon, Intervet). To recover mature oocytes, superovulated females were sacrified 15 hours post-hCG-injection. Oocytes were incubated after collection in 0.5% hyaluronidase (Sigma) in PBS for 1-2 min at 37° C. to remove cumulus. The total number of oocytes were counted.
[0112] Results
[0113] Vector Construction and Expression of sc-bFSH in
[0114] Although native bFSH is expressed from two different genes on different loci, we chose to genetically fuse the carboxyl end of the bFSH b subunit to the amino end of the a subunit according to Sugahara et al. (1996) in order to produce a single-chain bFSH (sc-bFSH). Both the receptor binding affinity and the potency of adenylate cyclase activation for the single-chain human FSH were shown to be similar to that of recombinant human FSH heterodimer (Sugahara et al. 1996). The fusion gene was inserted into the tobacco mosaic viral-based vector p4GD-PL (Casper and Holt 1996). In vitro run off transcripts of the construct p4GD-sc-bFSH were capable of infecting
[0115] 17-21 dpi infected leaves were harvested, total soluble protein (TO) extracted and subsequently subjected to SDS-PAGE. Silver staining revealed the abundant coat protein at position 17 kDa and a diffuse additional band at position 30 kDa, the expected size of undissociated alpha-beta bFSH heterodimer (Wu et al., 1992), which was not present in extracts of control plants. The corresponding Western blot analysis using an anti-humanFSH beta (hFSHβ) antiserum clearly confirmed the expression of the hormone by displaying a strong signal at position 30 kDa. Minor signals were obtained at 60 kDa which we interpret as artefactual dimerisation product that can occur during SDS-PAGE (Shi and Jackowski, 1998). Additionally, a putative degradation product of sc-bFSH was detected at approximately 15 kDa. It was not possible to reduce the amount of this degradation product by including a cocktail of plant protease inhibitors to the extraction buffer. The specificity of the signals obtained in sc-bFSH expressing plants was indicated by the absence of any signal in extracts from control plants.
[0116] Subcellular Localisation of sc-bFSH
[0117] In a series of experiments using different approaches we wanted to investigate whether the sc-bFSH as a glycoprotein hormone—in homology to the situation in mammals—is targeted to the secretory pathway of plant cells and if the protein is secreted into the periplasmic (extracellular) space. Ultracentrifugation of total protein extracts (TO) from sc-bFSH expressing plants was carried out which resulted in a pellet (P) and a supernatant (SN) fraction. All three fractions, TO, P and SN, were subsequently analysed by SDS-PAGE. The different staining patterns of SN and P on a silver-stained gel indicated a selective enrichment of soluble and of mostly membrane associated proteins, respectively. Silver staining and immunoblot analyses using anti-hFSHb antibodies clearly revealed the enrichment of the hormone in the soluble SN fraction, which is consistent with a periplasmic location.
[0118] The fact that no hormone -was detected in the P fraction excludes the formation of inclusion bodies which often is a consequence of protein overexpression.
[0119] As a next step a so-called intercellular washing fluid (IF), which is characterised by the specific enrichment of periplasmic (extracellular) proteins, was separated from the total protein extracts (TO) and compared with the remainder (RE) thereof. TO, IF and RE fractions were subjected to SDS-PAGE and silver staining revealed an additional diffuse band in the IF at the expected size of the hormone (30 kDa), which is absent in control IF fraction (
[0120] To confirm the periplasmic location of sc-bFSH, in situ indirect immunofluorescence was performed. Mechanical sectioning of epidermal cells of sc-bFSH expressing plants was used to provide entry sites for the antibodies. Hence, only cut cells show an immunostaining. The fluorescence signal was obtained in the periphery of the cells, being clearly different from a cytoplasmic or vesicular fluorescence staining as shown previously (Boevink et al., 1998; Essl et al., 1999). The in situ indirect immunostaining of sc-bFSH appeared as a thin film located inside of the cell wall being consistent with a periplasmic location.
[0121] N-glycosylation Analysis of sc-bFSH
[0122] Since native bFSH is a glycprotein hormone and its N-glycosylation is essential for bioactivity, the N-glycosylation status of sc-bFSH was investigated. Sc-bFSH has four potential N-glycosylation sites and, in Western blot analyses, a diffuse band was detected at position 30 kDa, which is larger than the expected size of the unglycosylated (23 kDa) protein (
[0123] The detailed structure of N-glycans attached to sc-bFSH was elucidated by MALDI-MS. This procedure was specially designed for the analysis of N-glycans potentially containing core fucose in a1,3 linkage. Immunoaffinity—chromatography using a monoclonal antibody against human FSH was carried out to purify the sc-bFSH from IF extracts, resulting in <<single band purity>> as evidenced by SDS-PAGE/silver staining. The sc-bFSH was subjected to tryptic in-gel digestion in order to provide susceptibility to the subsequent N-glycosidase A digestion. This further allowed the identification of the tryptic peptides measured by MALDI-MS (data not shown).
[0124] Subsequently, the enzymatically released N-glycans were cleaned up for MALDI-MS. The resulting mass revealed two peak masses that could be assigned to 2 known plant N-glycans of the paucimannosidic type: MMX and MMXF
[0125] FSH Receptor Activation by Plant sc-bFSH
[0126] To determine the in vitro bioactivity, the plant-expressed sc-bFSH was tested for its ability to induce cyclic AMP (cAMP) production in a CHO cell line expressing the porcine FSH receptor (pFSHR). Evidently, cAMP levels, as determined by RIA, were raised in a dose dependent manner upon addition of increasing amounts of pit-FSH. The effect of the plant expressed sc-bFSH on the production of cAMP in this cell line was determined by applying several nonpurified sc-bFSH-IF dilutions. Increasing concentrations of the sc-bFSH containing IF extract resulted in a dose responding cAMP production. The specificity of the cAMP production upon addition of plant-produced sc-bFSH was demonstrated by an absence of a response of the cells to an IF extract from control plants. The pit-bFSH standard curve allowed to estimate a concentration of 5 ng in vitro bioactive sc-bFSH per ml IF.
[0127] Example of an vivo Bioassay
[0128] Superovulatory Treatement of Mice
[0129] 15 6-8 week old female C57/CBA mice were treated each with 100 ml of 11 times concentrated sc-bFSH-IF extract (concentration was done using an AMICON ultrafiltration cell, MWCO 10 kDa). Serving as a negative control, 15 mice were treated each with 100 ml of 11 times concentrated IF-extract from not infected plants (NI-IF). Furthermore, the response of 14 mice to 5 IU pregnant mare serum gonadotropin (PMSG, Folligon, Intervet) in the same volume was investigated. 46-48 hours post FSH or wt-IF injections, the 3 groups were treated with 5 IU human chorionic gonadotropin (hCG; Chorulon, Intervet). To recover mature oocytes, superovulated females were sacrified 15 hours post-hCG-injection. Oocytes were incubated after collection in 0.5% hyaluronidase (Sigma) in PBS for 1-2 min at 37° C. to remove cumulus. The total number of oocytes were counted. The total number of oocytes indicated a high superovulatory response of the mice to PMSG, where as much as approximately 4 fold more oocytes were counted as compared to the negative control. A significant, albeit comparably low, superovulatory response to sc-bFSH (1,5 fold above the negative control) was found. The mean number of oocytes for each group, WT-IF, sc-bFSH-IF and PMSG, and the respective standard deviations are illustrated.
[0130] Embryo Isolation and Culture
[0131] Female mice were treated with intraperitoneal injection of pregnant mare serum gonadotropin (Folligon-intervet, 5 IU), sc-bFSH-IF extracts, WT-IF extracts, followed by human chorionic gonadotropin (chorulan, intervet) 48 h later. As a control, untreated females that showed an oestrus behaviour were included in this study. Mice were caged overnight with males and 1 cell stage embryos were isolated 19-26 hours after hCG from females showing sperm vaginal plugs (day 1). The ampullary regions of excised oviducts was placed at 30° C. in PBS medium containing bovine serum albumine at 4 mg/ml together with bovine hyaluronidase (sigma) at 50 units/ml. After 3-5 minutes the cumulus cells were dissociated and the eggs washed several times in PBS medium. Fertilized eggs showing two pronuclei and polar body were pooled from several females of the same group. Embryos were cultured under paraffin oil (DBH) in 10 μl drops of Whitten's medium in an atmosphere of 5% CO2 in air at 37° C.
[0132] Embryos were cultured for up to 72 hours in vitro in Whitten's medium and examined several times. For each group, development was assessed by the proportion of fused eggs that became blastocysts. The results clearly indicated that, whatever the treatment, more than 80% of the fused eggs reached the morula stage 3, 5 days after fecondation while at 7, 5 days more than 50% of them developed into bastocysts. Our results clearly showed no differences between PMSG and plant FSH extract treated females. These experiments strongly suggested that crude extract of infected plants containing FSH did not induce any deleterious effects on mice embryo further development, indicating potential use for assisted reproduction.
[0133] Example of Assays for Detecting the Presence of sc-bFSH-FSH Receptor Complexe
[0134] Two different assays were performed to detect the presence of sc-bFSH-FSH receptor complexe (FSHC) in tobacco leaves.
[0135] 1. Elisa
[0136] The wells of M96 microtiterplate were coated with polyclonal antiserum against FSH receptor (dilution at 1/200). To each coated well, 100 μl was added of serial dilutions of either WT-IF extract or intracellular fluid from infected tobacco leaves containing FSHC.
[0137] After incubation (1 h at 37° C.) and washing, a monoclonal antibody against human FSHβ (0.01 mg/ml) was added and incubated (1 h at 37° C.) which was followed by washing and addition of anti-mouse IgG coupled to peroxidase (1:500, 100 μl/well)
[0138] After washing, TMB and H
[0139] 2. Immunoradiometric Assay (IRMA)
[0140] The assay used coated beads for the capture of FSHC from infected tobacco leaves. Polystyrene beads were incubated overnight at 4° C. in the presence of polyclonal antibody against FSH receptor. After washing with distillated water, beads were used to capture antibody-reactive molecules present in the IF extract (WT or FSHC). After 2 h incubation at followed by extensive washing, labeled diluted monoclonal antibody against FSHP (in 0.01 M PiNacl containing 50% Foetal Calf Serum) was added. The reaction mixture was incubated for 1 h at 20° C., followed by a washing step and residual radioactivity was counted.
[0141] Here we demonstrated the rapid and high level expression of a single-chain version of the bovine follicle stimulating hormone (sc-bFSH) in
[0142] Most clinically important mammalian proteins, such as TSH, FSH and LH, have N-glycans, which confer different biological functions, such as resistance to protease attacks, antigenicity, immunogenicity and, as for FSH, plasma clearance rates. Although N-glycosylation is conserved in higher organisms to some extent, so far no established heterologous expression system produces correct mammalian-type N-glycans, due to more or less differing biosynthetic pathways. The perspective to use plants as economic factories to produce therapeutic recombinant proteins at a low cost makes it important to investigate the capacity of plant cells to produce functional mammalian-like glycoproteins. Our detailed analysis on the N-glycosylation pattern of sc-bFSH constitutes a complete study of a mammalian glycoprotein. Surprisingly, only two oligosaccharide structures were found N-linked in sc-bFSH and were identified as paucimannosidic N-glycans containing b1,2 xylose and a1,3 fucose residues in a ratio 80:20%, respectively. Paucimannosidic glycans are considered as typical vacuole-type N-glycans, which result from the elimination of the terminal residues of complex-type N-glycans in post-Golgi compartments (for review see Lerouge et al. 1998). Secreted proteins in plants usually carry complex-type N-glycans with a high degree of heterogeneity (Melo et al., 1997, Ogawa 1996, for a review see Sturm 1995). Although paucimannosidic N-glycans have been found to a minor extent in secreted proteins, the presence of this type of glycan as predominant species is a rather unusual case. Still, to our knowledge, there is only one detailed comparative study of a mammalian glycoprotein, a mouse immunglobulin ("plantibody"), produced in a plant expression system (Cabanes-Macheteau et al., 1999). In contrast to sc-bFSH the plantibody shows a higher degree of N-glycan heterogeneity, as a total of 8 different species of oligosaccharides were found. However, since no detailed analysis of the protein location was done it cannot be excluded that fractions of plantibodies are stored in different compartments of the plant cell (Cabanes-Macheteau et al., 1999). To our knowledge this is the first report of a detailed N-glycan analysis of a protein derived from an IF fraction, usually secreted proteins are analysed from total protein fractions. This might be a reason why less heterogeneity was found.
[0143] Evidently, the N-glycans present on sc-bFSH exhibit considerable structural aberration from its native counterpart, pituitary bFSH (Baenzinger and Green, 1988). As anticipated from known plant N-glycan structures, no N-glycans of the mammalian complex-type were found, neither b1,4 linked galactose nor terminal sialic acid. b(1,2) xylose and core a(1,3) fucose have never been found in mammals cells and they are considered potentially immunogenic structures (Wilson et al., 1998; Kurosoka et al., 1991; Faye et al., 1993??).
[0144] Although so far no negative effect has been reported for plantibodies applied to mammals which might result from these sugars, no long term studies are available.
[0145] We showed evidence, that the plant-produced hormone has in vivo bioactivity.
[0146] As appointed above, another important aspect of these experiment is the fact that the application of a highly concentrated IF extract, which comprises a complex mixture of periplasmic proteins, did not have an deleterious effect on the model animal. Unlike other established protein expression systems, such as bacterial, yeast or animal cell culture systems, plant IF extracts may be directly applicable in acute medical treatments without the need of further expensive purification.
[0147] In summary we conclude that the TMV-based expression system provided here gives a very attractive expression system for mammalian glycoproteins such as glycoprotein hormones, since bioactive glycoforms of sc-bFSH accumulate to high levels in the periplasmic space of
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Susan had her web developer set up Google AdWords Anomaly Alerts so she would know when her AdWords account was underperforming. Shortly after going live with the new code, Susan received an alert. She opened the email and learned that she had an anomaly; but to her frustration, that was all the alert told her. It did not tell her what part of her account had generated the alert, nor did it tell her what outside influences might be causing the decreased performance.
13 tips to make your social media posts better
1. Use rich media to get more attention
Share images and videos of your products or people using your products will make you more engaged which will generate more attention for your services. Keep your posts concise and to the point to maximize your level of engagement with new and loyal customers.
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4 Ways to Improve Your Mobile Marketing
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How Tra-Digital Advertising is the Future of All Advertising
The traditional advertising realm was fundamentally altered when the boom of digital advertising came about in the early 2000's.
It shook the fabric of the status quo and literally split the marketing world into two different but equally important segments. Over the course of the last 15 years, traditional agencies have always struggled grasping and delivering high-quality digital campaigns. Inversely, digital agencies have had an equally difficult time breaking into the traditional world. This unfortunate trend has been at the expense of the consumer, until now.
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Google Brings in a New Data Distribution Module to Adwords – Attribution 360
Google recently announced an upcoming data- driven attribution model to AdWords. The change means that users can now allocate an attribution model to every conversion type that has been established in AdWords. As a result, nobody will be required to use last-click as a default setup for assigning conversion credits.
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Facebook: Changing the Way We Drive Leads
Facebook lead ads give advertisers the opportunity to gather information and leads directly from mobile ads. It bypasses the need to get users to click-through, fill out a form and SUBMIT. Instead, in Facebook, once a user clicks on an ad, information like name and email will be pre-populated. You can set up customized form fields for accumulating information that fit business needs.
How Does This Work?
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subset: commoncrawl


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World's First RISC V based FPGA SoC Brings An Open Source Ecosystem With It
RISC-V development solutions are finally coming to market, with the world's first RISC-V-based SoC released by Microsemi, a Microchip Company. Tim Morin, the company's Director of Product Marketing for Programmable Solutions discusses the architecture of the FPGA SoC, and how integrations with Antmicro's Renode software development framework and Mi-Five Ecosystem are helping engineers bring open-source hardware and software solutions to market quickly, and how THEY choose. Brandon Lewis, Editor in Chief of Embedded Computing reporting.


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Botanical Collages by Lisa Dawn White
These beautiful handmade botanical collages are entirely handmade of pressed botanicals and handmade painted paper. Each is one-of-a-kind and uniquely designed to withstand light and humidity. Starting at $75 for a 5×5 size.
Because each piece is one-of-a-kind, please call the gallery to find out which pieces are currently available at 1-844-234-4387.


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- No Fruity Pebbles in mom and dad's bed(what a ginormous mess that is to clean!)
- Stop watching Mighty Beanz computer videos long enough to go the bathroom when you need to!
- Say your "please" and "thank you's"
and so on and so forth...
One of these life lessons has been the very simple (yet so very important to me) lesson of doing things for others and the reward of simply seeing the joy it brings to them. I can still hear my own mother telling me as a child, "Jan, the whole world does not revolve solely around you!" A bit harsh maybe, but it has served me well in my life thus far. I only wish I had lived it a bit more fully in my twenties. My hope and prayer is that my boys will understand this early on and have a desire to think beyond just themselves.
So, I thought it would be a fun little exercise for us to suprise his cousins with a little gift...as in a suprise "egging" of their house. That being "egging" in a good way not the heathen approach taken previously by
Pierce and I agreed that Flaming hot cheetos was the only way to go for the less conventional easter carrots.
Only the very best for family.
The happiness it brought to Pierce's face to get the much anticipated phone call from his cousins that the suprise was, indeed, a suprise, was so much fun! He was so proud and took full ownership of his handywork.
Of course once the phone call ended, he was quick to ask when or if this suprise would be reciprocated....Baby steps, I say.
Want to suprise someone today? Doesn't have to be a fancy or huge production. A simple dollar store basket and some sweet treats could be all it takes to make someone's day. Head over here to get your too cute "You've been egged!" proclaimation.


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Bootcamp SKEMA Ventures 2019: Fast track your startup's progress
Ahoy, student entrepreneurs! If you are looking to strengthen your entrepreneurial venture, SKEMA Ventures is bringing to you the perfect opportunity: the exclusive three-day Bootcamp!
The Bootcamp is an intensive event that trains the participants in the process of formulating the framework of their startup, fine-tuning the business plan, understanding its uses for managing the launch of the new venture, and attracting outside investors.
This event is for entrepreneurs who are working on an innovative project and are willing to prepare to present it in front of potential partners, investors and clients. The focus is on helping participants to move forward and acquire their first clients as soon as possible. The selected entrepreneurs will get to collaborate with coaches to design/fine-tune their business plan and pitch it in front of an audience of professionals (entrepreneurs, investors, faculty, and so on).
The event will involve three days of knowledge transfer and iterative practice, coaching, keynotes from inspiring entrepreneurs, training for pitching, and more!
The Bootcamp is being held from May 13 to 15 at SKEMA Business School, Paris campus, Pole Universitaire Léonard de Vinci, La Défense. The projects will be accepted on first come, first serve basis, so register as soon as possible and block these dates!
The deadline to apply is May 8.
The process to register is simple. Please register at https://gust.com/programs/svf_bootcamp.
We look forward to seeing you at the Bootcamp!
Skema Ventures View All
SKEMA Ventures is a business unit created by SKEMA Business School dedicated to entrepreneurship and innovation. Through a unique value chain, that encompasses teaching, coaching, incubation, and acceleration, SKEMA Ventures allows each SKEMA student and alumni to think, design, test and launch an entrepreneurial project in a global context, on six innovative territories on four continents, benefiting from the best of each local ecosystem. With SKEMA Ventures, GloCal entrepreneurship is born!
Watch this video to know more: https://youtu.be/OAYVo9wtWZM


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Read our blog for the latest insights on sales and marketing Take Me There
Webinar: Document Merge in wMobile Desktop for GoldMine Register
SugarCRM's next product release, scheduled for April 27th, is known as Sugar 8. This release includes all the features from the last two product releases and Spring '18 which will also come out on April 27th. Here's a sneak peek of the features new to on-premise users in this update.
Sugar 8 includes a new user interface that is cleaner, more modern, and more consistent. While the switch from Sugar 6 to Sugar 7 was a challenge for some companies, the upgrade to Sugar 8 should be more straightforward merely because these changes are visual updates only to HTML and CSS. The new appearance is more intuitive than ever and now matches the mobile app design for consistency across devices.
The new Product Catalog Dashlet lets users click on items within a product catalog and quickly add them to a quote or other documents that they generate in Sugar. The product catalog is a visual representation of all your products, available within the intelligence panel.
Another reporting improvement! Users can click on a segment of a chart or graph and see a drill down of the data within that segment. Once users are in the list view, they can modify data using in-line editing.
The old email client (did anyone even use that?) has been deleted and replaced with a much-improved emails module. The new module is built on the sidecar framework (coding speak for the fancy side panel) and allows you to preview emails from subpanels or list views by clicking on the eye icon.
Email Sub-Panel in Record View
Email Preview in Intelligence Panel
The Sugar 8 release will also include several brand new features from the coming Spring '18 release. The details of this release haven't gone public yet--so we can't spill the beans (or the sugar cubes). Stay tuned on April 27th for a release recap.
P.S. In late 2017 Sugar updated their release schedule--get the scoop on that here. Since then, on-demand users see a product release once per quarter, and on-premise users get a roll-up release once per year.


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Here's a question: If you could fly a spacecraft from one end of the galaxy all the way to the other, in a straight line, what are the chances you'd make it through without hitting anything?
That's the scenario Reddit user bigri23 put forth on the "Ask Science" subreddit. The answer might surprise you: It's pretty close to 100 percent. In other words, your likelihood of hitting something is zero.
Fellow Redditor astrocubs has the top answer, which explains why the chances of running into something on a flight through the universe are actually very, very small. (This is assuming that "something" is bigger than a pebble, which rules out gas and dust, and only considers something planet-sized or bigger). Here's how astrocubs got there:
Around the Sun, there are about 0.004 stars per cubic light year. Near the center of the galaxy this will be higher, and at the edges it will be lower. To be conservative and try to hit something, let's just say the galactic average is 1000x what it is near the sun: 4 stars / cubic light year. (NOTE that this is pretty absurd.)
The vast majority of stars are smaller than the Sun, but a tiny percentage are way, way bigger. Calling them all Solar-radius might be a decent estimate. But let's go crazy and say an average star has 100x the Sun's radius. (Again, this is absurd, but we're trying to hit something.)
We can ignore planets, black holes, neutron stars, etc. because their cross-sectional areas are just minor corrections to the stars. E.g. the cross-sectional area of all planets in the solar system would just be adding 3 percent to the Sun.
With all that in mind, and all those assumptions—even the ones that exaggerate our chances of hitting something—in place, astrocubs estimates that our theoretical spaceship would have to fly from one end of the galaxy to the other 15,000 times before ever running into anything.
Or, as astrocubs puts it, "you have a 0 percent chance (within rounding errors)" of encountering another object during that trip.
I asked Matthew Francis, a physicist and science writer, whether the answer astrocubs provided was reasonable. Without crunching the numbers himself, he said that it was likely right. "If you don't count tiny stuff like gas particles, the galaxy is a pretty empty place!" he told me in an email. "In fact, when two galaxies collide, the chances of individual stars running into each other are slim, even though the galaxies themselves get all stretched out of shape. If you got in a spaceship and went from one side of the galaxy to the other (assuming you didn't aim right at the black hole at the center), you'd probably miss everything."
The calculations here are firmly in the world of theory, and don't take into account the actual positions of planets or stars, or the existence of the space junk that we humans are constantly leaving behind. But even though the amount of trash we've deposited is dangerous and ugly, it's tiny compared the size of the universe.
Yet another reminder that space, for all its wonders and marvels, is a very empty place.
We want to hear what you think about this article. Submit a letter to the editor or write to firstname.lastname@example.com.


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If your new year's resolution to get fit has gone down the pan (we gave up trying to get fit on January 3rd, FYI), why not try a slightly more fun form of gym? Tate Liverpool's new Art Gym series has nothing to do with toning your calves and everything to do with toning your creativity instead. Now that's more like it.
Art Gym's offering everyone, regardless of age or background, the ability to create a personalised programme of skill exploration – from bookmaking to digital art, it's a lot more enjoyable than sweating out a hangover on a treadmill. Turner Prize winning collective Assemble, who did a sterling job of Granby Street, have been called in to create the custom-built Art Gym space which will hold workshops, lectures, and classes throughout March with a series of creative bright sparks. All the satisfaction of a workout without the need for lycra or Lucozade. Perfect.
Art Gym at Tate Liverpool
7 – 31 March


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Welcome to Peopleware India
Peopleware India specializes in providing advance/Level 400+ learning solutions on Microsoft & Non-Microsoft Products & Technologies. We undertake corporate training assignments in India and across the globe. We also organize calendar training programs for our customer across India. We are preferred vendors to some of the largest companies in India. Our esteemed clientele includes Microsoft, Wipro, Infosys, Accenture, Intel, HCL, Hexaware, US Technology, Siemens, RBS, Adobe & many other IT & non-IT giants. We are Microsoft Partners and an ISO 9001:2000 company.
We have Microsoft Practice comprising of a pool of technology trainers on .NET, SharePoint, BizTalk, SQL Server, Azure, Office, Windows Infrastructurem Exchange and a couple of other server products, etc. Peopleware India is led by Amit Bansal (www.amitbansal.net) who specializes in SQL Server & Business Intelligence and have delivered more than 300 workshops/courses across the globe with 100+ clients. He is also a MVP on SQL Server (Microsoft Most Valuable Professional).
Why Peopleware India?
- Specialised, advance technical training on Microsoft & non-Microsoft products & technologies – we focus on Level 400+ assignments
- Some of the best technology trainers, consultants & mentors on board, delivering the most challenging assignments through out the world serving more than 300+ clients
- Peopleware India is lead by Amit Bansal, a veteran in SQL Server & Business Intelligence – most of the advance trainers are hand-picked by him
- Esteemed clientele includes Microsoft, Infosys, Wipro, Accenture, CapGemini, RBS, HCL, Hewitt Technologies, Hexaware, Robert Bosch, TATA Interactive Systems, Siemens, US Technology, Hewlett Packard, IBM, LexMark, GlobalLogic and many other CMM Level 5 companies.
- Peopleware India's commitment to quality & its business ethics & integrity is unmatched.
- Well-organized and widely accepted course catalog
- More than 2000+ workshops delivered - Browse some of them at our Gallery page
OUR MISSION: To conduct and successfully deliver courses and training programs on cutting edge technologies by top notch trainers, in a corporate learning environment to create highly skilled workforce.
OUR VISION: To be recognised globally as a team of specialists in technology training.