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Ali has had music in her life since she was in the crib. She has performed professionally in the musical theatre world and she has studied in several universities for Musical Theater, Jazz, Pop, Blues, and R&B. Since the age of sixteen, she has been selected to sing as a guest soloist for venues such as Spivey Hall, Georgia World Congress Center, and Georgia Tech Hotel Conference Center. Ali has also sung for many movies and recordings, and recently recorded a demo for David Foster, Barbra Streisand, and Andrea Bocelli and recently toured as a pianist for "The Women of Ireland." She is a recent graduate of Berklee College of Music and earned her BM in Music Business and Vocal Performance cum laude studying under Magi Scott, Jeff Ramsey, Jerome Kyles, and Carolyn Escoffrey, Donna McElroy,
My name is Justin P. I have a love for music that started when I was two years old when I would always want to play on any piano, or keyboard that was around. At the age of five years old, my parents signed me up for piano where I played for 13 years at the The Piano Academy in College Park, Georgia. At the age of ten years old, I joined the band at Fayette Middle School where I started playing percussion. Upon graduation from high school and the Piano Academy, I attended the Atlanta Institute of Music where I studied in a two year rigorous musicianship program. I completed the program with a Certificate of Musicianship in Music Production, Engineering and Recording. *** Travel Equipment *** I will provide the music. Students must provide a piano *** Specialties *** Piano
With studies at Queens College NYC, the Juilliard School and the Brooklyn Conservatory Of Music, you are assured of a first class educational experience. When you couple that with someone that has been on the road with some of the top names in the music business and knows their way around a high tech recording studio environment, you get a very unique and exciting lesson experience. Kenny Keys , a name given to him by one of his former employers rap mogul Russell Simmons, is a dynamic, creative and well rounded professional musician with over 20 years of teaching experience. Growing in Queens NYC and later moving to VA Beach VA and now a resident in the ATL area, Kenny Keys has work in almost every genre of the music industry from Gospel, R&B, Jazz to Hip Hop. "I am well versed in Music
I love playing the guitar. I think you will too when you see the results that come from dedication and motivation. Everyone who plays the guitar is a student of the instrument. The greatest players in the world are still learning. If you are a beginner or an advanced player there is always room for improvement. No matter your skill level, I can help you grow in knowledge and technique. *** Lesson Details *** I can teach you in the styles of country, blues, folk, rock, pop, and jazz. Music is organized sound. I will teach you how music works and how to apply that knowledge to the guitar. Trust me, music theory is NOT complicated. You do not have to read music to understand music theory. Within a few months if you dedicate plenty of time and effort to the lesson materials you ...
I am a professional guitarist/bassist with more than 20 years experience playing in bands of various styles including hard rock, metal, country and jazz. I have toured across the U.S. and have shared the stage with acts as varied as Styx, Heart, Foreigner, Corey Smith, Drivin' & Cryin', Seven Mary Three, and Quiet Riot. *** Lesson Details *** My students can expect their lessons to be laid back but still goal oriented. I strive to tailor lessons to the personal learning styles and musical preferences of my students. If the student wants to be the next solo-guitar shredder or introspective guitar songwriter, I want to give him/her the tools needed to succeed. After just a few months (with consistent practice at home), my students will be able to not only play along with their favorite ...
Music is my passion, my hobby, my career and my gift. I have been performing since age 7 and teaching for the last 25 years. I have a bachelor's and master's degree in music and love to share my knowledge and experience with all ages and aptitudes - even special needs. My approach to teaching is fun but serious, laid-back but encouraging, demanding but forgiving. I teach students how to be disciplined with their practice and how to make the most of their limited time. *** Lesson Details *** I can help you achieve your goals! For those who want a well-rounded music education, I include music theory (note and rhythm reading), technique, ear training, improvisation and performance practice in my curriculum using a variety of methods. I enjoy working with adults interested in ...
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Methods for enhancing expression levels and secretion of heterologous fusion proteins in a host cell are disclosed.
1. A kit comprising a recombinant vector containing a nucleic acid sequence encoding small ubiquitin related modifier (SUMO) operably linked to a promoter and a multiple cloning site; wherein said multiple cloning site allows for inserting a nucleic acid encoding a protein of interest in-frame and immediately 3′ to the nucleic acid sequence encoding the Gly-Gly cleavage site of SUMO.
2. The kit of
3. The kit of
4. The kit of
5. The kit of
6. A kit for purification of a protein from a host cell comprising:
i) a recombinant vector comprising:
a) a nucleic acid sequence encoding SUMO;
b) a promoter;
c) a nucleic acid sequence encoding for a purification tag; and
d) a multiple cloning site;
wherein said promoter is operably linked to said nucleic acid sequence encoding SUMO, wherein said nucleic acid sequence encoding a purification tag is in-frame and operably linked to the nucleic acid sequence encoding SUMO, and wherein said multiple cloning site allows for inserting a nucleic acid encoding a protein of interest in-frame and immediately 3′ to the nucleic acid sequence encoding the Gly-Gly cleavage site of SUMO, and
ii) a composition comprising a protease which specifically cleaves SUMO after the Gly-Gly cleavage site.
7. The kit of
8. The kit of
9. The kit of
i) a solid support for binding the purification tag,
ii) lysis buffers,
iii) wash buffers,
iv) elution buffers,
v) cleavage buffers, and
vi) instruction material.
10. The kit of
11. The kit of
12. The kit of
13. The kit of
14. The kit of
15. The kit of
This application is a continuation-in-part of U.S. application Ser. No. 10/338,411 filed Jan. 7, 2003, issued as U.S. Pat. No. 7,060,461, which claims priority to U.S. Provisional Application No. 60/346,449 entitled �Methods for Protein Expression and Purification� filed Jan. 7, 2002.
The present invention relates to the field of recombinant gene expression and purification of expressed proteins. More specifically, the invention provides materials and methods which facilitate purification of heterologous proteins from a variety of different host species.
Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Full citations for those references that are numbered can be found at the end of the specification. Each citation is incorporated herein as though set forth in full.
Functional genomic studies have been hampered by the inability to uniformly express and purify biologically active proteins in heterologous expression systems. Despite the use of identical transcriptional and translational signals in a given expression vector, expressed protein levels have been observed to vary dramatically (5, 7). For this reason, several strategies have been developed to express heterologous proteins in bacteria, yeast, mammalian and insect cells as gene-fusions.
The expression of heterologous genes in bacteria is by far the simplest and most inexpensive means available for research or commercial purposes. However, some heterologous gene products fail to attain their correct three-dimensional conformation in E. coli while others become sequestered in large insoluble aggregates or �inclusion bodies� when overproduced. Major denaturant-induced solubilization methods followed by removal of the denaturant under conditions that favor refolding are often required to produce a reasonable yield of the recombinant protein. Selection of ORFs for structural genomics projects has also shown that only about 20% of the genes expressed in E. coli render proteins that were soluble or correctly folded (36, 38). These numbers are startlingly disappointing especially given that most scientists rely on E. coli for initial attempts to express gene products. Several gene fusion systems such as NUS A, maltose binding protein (MBP), glutathione S transferase (GST), and thioredoxin (TRX) have been developed (17). All of these systems have certain drawbacks, ranging from inefficient expression to inconsistent cleavage from desired structure. Comprehensive data showing that a particular fusion is best for a certain family of proteins is not available.
Ubiquitin and ubiquitin like proteins (UBLs) have been described in the literature. The SUMO system has also been characterized. SUMO (small ubiquitin related modifier) is also known as Sentrin, SMT3, PIC1, GMP1 and UBL1. SUMO and the SUMO pathway are present throughout the eukaryotic kingdom and the proteins are highly conserved from yeast to humans (12, 15, 28). SUMO homologues have also been identified in C. elegans and plants. SUMO has 18% sequence identity with ubiquitin (28, 39). Yeast has only a single SUMO gene, which has also been termed SMT3 (23, 16). The yeast Smt3 gene is essential for viability (29). In contrast to yeast, three members of SUMO have been described in vertebrates: SUMO-1 and close homologues SUMO-2 and SUMO-3. Human SUMO-1, a 101 amino-acid polypeptide, shares 50% sequence identity with human SUMO-1/SUMO-2 (29). Yeast SUMO (SMT3) shares 47% sequence identity with mammalian SUMO-1. Although overall sequence homology between ubiquitin and SUMO is only 18%, structure determination by nuclear magnetic resonance (NMR) reveals that the two proteins share a common three dimensional structure that is characterized by a tightly packed globular fold with β-sheets wrapped around one α-helix (4). Examination of the chaperoning properties of SUMO reveals that attachment of a tightly packed globular structure to N-termini of proteins can act as nucleus for folding and protect the labile protein. All SUMO genes encode precursor proteins with a short C-terminal sequence that extends from the conserved C-terminal Gly-Gly motif. The extension sequence, 2�12 amino acids in length, is different in all cases. Cells contain potent SUMO proteases that remove the C-terminal extensions. The C-terminus of SUMO is conjugated to E amino groups of lysine residues of target proteins. The similarity of the enzymes of the sumoylation pathway to ubiquitin pathway enzymes is remarkable, given the different effects of these two protein modification pathways. Sumoylation of cellular proteins has been proposed to regulate nuclear transport, signal transduction, stress response, and cell cycle progression (29). It is very likely that SUMO chaperones translocation of proteins among various cell compartments, however, the precise mechanistic details of this function of SUMO are not known.
Other fusions promote solubility of partner proteins presumably due to their large size (e.g., NUS A). Fusion of proteins with glutathione S-transferase (GST) or maltose binding protein (MBP) has been proposed to enhance expression and yield of fusion partners. However, enhanced expression is not always observed when GST is used as GST forms dimers and can retard protein solubility. Another problem with GST or other fusion systems is that the desired protein may have to be removed from the fusion. To circumvent this problem, protease sites, such as factor X, thrombin or Tev protease sites are often engineered downstream of the fusion partner. However, incomplete cleavage and inappropriate cleavage within the fusion protein is often observed. The present invention circumvents these problems.
In accordance with the present invention compositions and methods for enhancing expression levels of a protein of interest in a host cell are provided. An exemplary method comprises i) operably linking a nucleic acid sequence encoding molecule selected from the group consisting of SUMO, RUB, HUB, APG8, APG12, URM1, and ISG15 to a nucleic acid sequence encoding said protein of interest thereby generating a construct encoding a fusion protein, ii) introducing said nucleic acid into said host cell, whereby the presence of said molecule in said fusion protein increases the expression level of said protein of interest in said host cell. In a preferred embodiment the molecule is SUMO encoded by a nucleic acid of SEQ ID NO: 2. The method optionally entails cleavage of said fusion protein and isolation of the protein of interest.
In yet another embodiment of the invention, an exemplary method for generating a protein of interest having an altered amino terminus is provided. Such a method comprises i) providing a nucleic acid sequence encoding the protein of interest; ii) altering the N-terminal amino acid coding sequence in the nucleic acid; iii) operably linking a SUMO molecule to the nucleic acid sequence; and iv) expressing the nucleic acid in a eukaryotic cell, thereby producing the protein of interest in the cell, wherein the eukaryotic cell expresses endogenous SUMO cleaving enzymes, which effect cleavage of SUMO from the sequence encoding the protein of interest, thereby producing a protein of interest having an altered amino terminus. All amino acids with the exception of proline may be added to the amino terminus using this method.
The invention also provides a method for producing a sumolated protein for tracking protein localization within a host cell. An exemplary method comprises i) providing a nucleic acid sequence encoding said protein; ii) substituting the N-terminal amino acid coding sequence in the nucleic acid for a codon which encodes proline; iii) operably linking a SUMO molecule to said nucleic acid sequence; and iv) expressing said SUMO linked protein in said host cell.
In another aspect of the invention, a method for enhancing secretion levels of a protein of interest from a host cell is provided. Such a method comprises i) operably linking a nucleic acid sequence encoding molecule selected from the group consisting of SUMO, RUB, HUB, URM1, and ISG15 to a nucleic acid sequence encoding said protein of interest thereby generating a construct encoding a fusion protein, ii) introducing said nucleic acid into said host cell, whereby the presence of said molecule in said fusion protein increases the secretion of said protein of interest from said host cell.
In yet a further aspect of the invention, kits are provided for performing the methods described above. Such kits comprise a recombinant vector containing a nucleic acid sequence encoding a UBL molecule selected from the group of SUMO, RUB, HUB, URMl, and ISGl5 operably linked to a promoter suitable for expression in the desired host cell and a multiple cloning site suitable for cloning a nucleic acid encoding the protein of interest. The recombinant vector may also contain a nucleic acid sequence encoding for a purification tag. The kits may further comprise a preparation of a protease capable of cleaving the UBL molecule from the fusion protein, an appropriate solid phase for binding the purification tag, appropriate buffers including wash and cleavage buffers, and frozen stocks of host cells. The host cells may be selected from the group of yeast cells, E. coli, insect cells, and mammalian cells.
There are a number of reasons for the lack of efficient recombinant protein expression in a host, including, for example, short half life, improper folding or compartmentalization and codon bias. While the Human Genome project has successfully created a DNA �map� of the human genome, the development of protein expression technologies that function uniformly in different expression platforms and for all the protein motifs has not yet been achieved.
In accordance with the present invention, it has been discovered that that N-terminal fusion of the ubiquitin homologue SUMO or Smt3 to otherwise unexpressed or poorly expressed proteins remarkably enhances the expression levels of biologically active proteins in both prokaryotes and eukaryotes. The Ubiquitin-Like protein (UBL) family contains many proteins, including for example, SUMO, Rub1, Hub1, ISG15, Apg12, Apg8, Urm1, Ana 1a and Ana 1b (15, 28). See Table 1. The hallmark of all of these proteins, exept APG12, and URM1, is that they are synthesized as precursors and processed by a hydrolase (or proteases) to generate mature carboxy-terminal sequence. Secondly, all of the UBLs share a common structure.
In E. coli, fusion proteins remained intact while in yeast or insect cells fusion proteins were efficiently cleaved, except when proline was the N-terminal residue of the target protein. While any of the UBLs set forth in Table 1 may be utilized in the compositions and methods of the invention to enhance expression of heterologous fusion proteins of interest, SUMO is exemplified in the gene fusion system provided herein.
The SUMO fusion system of the present invention has been successfully applied to express different molecular weight proteins such as 6 KDa Protein G domain to 110 KDa β-galactosidase in E. coli and eukaryotic cells. More specifically, the system allows one to: (1) enhance the expression of under-expressed proteins; (2) increase the solubility of proteins that are insoluble; (3) protect candidate proteins from degradation by intracellular proteases by fusing UBLs to their N-termini; (4) cleave the fusion protein to efficiently generate authentic proteins using naturally-present enzymes (5) generate proteins with novel amino termini; and (6) cleave all fusion proteins with remarkable efficiency irrespective of the N-terminal sequence of the fused protein, using UBL hydrolases such as SUMO hydrolase Ulp1. Because UBLs are small molecular weight proteins (�100 amino acids), they can also be used as purification tags as well. These remarkable properties of UBLs make them excellent candidates for enhancing expression and solubility of proteins. The method may also be utilized to generate novel amino termini on proteins of interest for a variety of research, diagnostic and therapeutic applications.
The ultimate fate of ubiquitinated or sumoylated proteins within a cell varies. A protein can be monoubiquitinated or polyubiquitinated. Ubiquitination of protein has multiple functions and gives rise to different fates for the protein within a cell (11). Ubiquitination primarily targets proteins to 26S proteosome for degradation (13). On the other hand, sumoylation of target proteins does not lead to degradation, but, rather, leads directly or indirectly to altered localization of proteins (15). There are about 17 deubiquitinating enzymes that cleave conjugated ubiquitin from target proteins as well as ubiquitin-ubiquitin and ubiquitin artificial-fusion proteins (1, 35). Thus far it appears that yeast has two cysteinyl proteases, called Ulp1 and Ulp2, that remove SUMO from ε-amino groups of lysine as well from the artificial linear SUMO-fusions (20, 21).
To determine if UBLs and SUMO fusion will enhance expression of recombinant proteins of different sizes and function, we have designed several UBL-GFP fusion proteins in addition to SUMO-fusion proteins and monitored their expression levels in E. coli, yeast and insect cells. In E. coli, the proteins are expressed as intact fusions, while in eukaryotes, the fusions were efficiently cleaved. A dramatic increase in the yield of proteins after fusion with SUMO and expression in E. coli was observed. In additional studies, SUMO-GFP protein was used as a model fusion for detailed studies in yeast and insect cells. We have designed SUMO-GFP fusion where all the N-terminal methionine residues have been replaced with the rest of the 19 amino acids. We have purified 20 sumo-GFP fusion proteins from E. coli and cleaved them in vitro with Ulp1. Ulp1 efficiently cleaved 19 out of the 20 possible amino acid junctions. The proline junction was not cleaved. As compared to deubiquitinating enzyme (3), Ulp1 demonstrated broad specificity and robustness in its digestion properties. Proteins having a wide range of molecular weights were cleaved efficiently by Ulp1. Similarly, in yeast, and insect cells, the fusion proteins were efficiently processed, yielding intact, biologically active proteins. In addition to enhancing protein expression levels, the SUMO-fusion approach can be used to advantage to generate desired N-termini to study novel N-terminal protein functions in the cell. Since SUMO fusion can both enhance recombinant protein yield and generate new N-termini, this technology provides an important tool for post-genomic biotechnology analyses.
The present invention also encompasses kits for use in effecting enhanced expression, secretion, purification, localization, and alteration of the amino terminus of a protein of interest. Such kits comprise a recombinant vector containing a nucleic acid sequence encoding a UBL molecule selected from the group of SUMO, RUB, HUB, URM1, and ISG15 operably linked to a promoter suitable for expression in the desired host cell and a multiple cloning site suitable for cloning a nucleic acid encoding the protein of interest in-frame with the nucleic acid sequence encoding the UBL molecule. The promoter is preferably a strong promoter and may be constitutive or regulated. Such promoters are well known in the art and include, but are not limited to, the promoters provided hereinbelow such as the ADH1, T7, and CUP1 promoters.
The recombinant vector may also contain a nucleic acid sequence encoding a purification tag in-frame with the sequence encoding the UBL molecule. Purification tags are well known in the art (see Sambrook et al., 2001, Molecular Cloning, Cold Spring Harbor Laboratory) and include, but are not limited to: polyhistidine, glutathione-S-transferase, maltose binding protein, thioredoxin, the FLAG� epitope, and the c-myc epitope. Materials and methods for the purification of fusion proteins via purification tags are also well known in the art (see Sambrook et al., Novagen catalog, 2002, examples hereinbelow). Reagents including, but not limited to, solid supports capable of binding the purification tag, lysis buffers, wash buffers, and elution buffers may also be included in the kits.
The kits may further comprise a composition comprising a protease or proteases capable of cleaving the UBL molecule from the fusion protein, cleavage buffers, frozen stocks of host cells, and instruction manuals. The kits may also further comprise reagents for altering the nucleic acid encoding a protein of interest to generate amino termini which are different from those native to the wild-type protein. Methods for altering the nucleic acid are well known in the art and include, but are not limited to, site-directed mutagenesis and oligonucleotide-based site-directed mutagenesis (see BD Biosciences Catalog, 2001; Qiagen Catalog, 2001; Ausubel et al., eds., 1995, Current Protocols in Molecular Biology, John Wiley and Sons, Inc.).
As used herein, an �instructional material� includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition of the invention for performing a method of the invention. The instructional material of the kit of the invention can, for example, be affixed to a container which contains a kit of the invention to be shipped together with a container which contains the kit. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and kit be used cooperatively by the recipient.
The materials and methods set forth below are provided to facilitate the practice of the present invention.
Design and Construction of E. coli Expression Vectors:
The original vector backbone was developed using pET 24d vector from Novagen (see
Construction of Variable His6SUMO-GFP Fusions:
A N-terminal six his-tagged SUMO (fusion vector was constructed as follows. A PCR product was generated with the primers 5 ′ CCATGGGTCATCACCATCATCATCACGGGTCGGACTCAGAAGTCAATCAA- 3 ′ (SEQ ID NO: 40) and 5′-GGATCCGGTCTCAACCTCCAATC TGTTCGCGGTGAG-3′(SEQ ID NO:41) using yeast Smt3 gene (16) as a template (kind gift of Erica Johnson). The FCR fragment was double digested with Nco I and Bam HI, and then ligated into pET24d, which had been similarly digested. It is important to note that the current invention utilizes a variant of the wild type yeast SUIvIO sequence. The A nucleotide at position 255 has been replaced with a G nucleotide, thus encoding an alanine instead of a threonine (SEQ ID NOS: 64 and 65). The detailed cloning strategy is provided in
Construction of SUMO-Fusion Vectors from pSUMO:
The gene encoding the protein of interest is cloned in frame with the SUMO tag, in the pSUMO vector, by utilizing the encoded Bsa I site. Bsa I belongs to the family of Class IIS restriction enzymes, which recognize non-palindromic sequences, and cleave at a site that is separate from their recognition sequences. The latter trait gives Class IIS enzymes two useful properties. First, when a Class IIS enzyme recognition site is engineered at the end of a primer, the site is cleaved when digested. Second, overhangs created by Class IIS enzymes are template-derived and thus unique. This is in clear contrast to regular Class II restriction enzymes such as EcoRI, which creates an enzyme-defined overhang that will ligate to any EcoRI-digested end. The unique overhangs produced by Class IIS enzymes can be ligated only to their original partner.
It is often preferable to amplify the gene encoding the protein of interest via PCR prior to cloning into the pSUMO vector. The forward primer must contain the additional standard sequence:
5′-GGTCTCAAGGTNNN-3′ (SEQ ID NO:44) where GGTCTC is the Bsa I site and NNN is the first codon of the gene encoding the protein of interest. Additional nucleotides are required for the primer to anneal specifically with the gene of interest during the PCR amplification. The reverse primer may contain another restriction enzyme such as Xho I to allow for directional cloning of a gene into pSUMO. Bsa I can also be employed in the reverse primer to simplify cloning steps, for example, in the following primer:
The B2 IgG binding domain (9) from streptococcus G148 protein was synthesized by three synthetic oligonucleotides. The sequence of the gene is 5′-GT CTTAAGA CTA AGA GGT GGC ACG CCG GCG GTG ACC ACC TAT AAA CTG GTG ATT AAC GGC AAA ACC CTG AAA GGC GAA ACC ACC-3′. (SEQ ID NO:46) The 81 bps oligo sequence is 5′-GCC GTT ATC GTT CGC ATA CTG TTT AAA CGC TTT TTC CGC GGT TTC CGC ATC CAC CGC TTT GGT GGT TTC GCC TTT CAG-3′. (SEQ ID NO:47) The 86 pbs oligo sequence is 5′-CAG TAT GCG AAC GAT AAC GGC GTG GAT GGC GTG TGG ACC TAT GAT GAT GCG ACC AAA ACC TTT ACC GTG ACC GAA TAA GGT ACC CC-3′ (SEQ ID NO:48). The bolded nucleotides refer to the AflII and Kpn1 sites that flank the protein G domain. ACG is the first amino acid residue of the domain. The above three oligos were annealed using the Life Technologies protocol. The annealed fragments were extended by Poll enzyme. The resultant gene was PCR amplified by the following oligo primers GI forward 5′-CTT GTC TTA AGA GGT-3′ (SEQ ID NO:49) and G2 reverse primer 5′-GCT GGG TAC CTT ATT CGG TCA-3′ (SEQ ID NO:50). The above protein G gene was cloned at the AflII and Kpn1 site of the human ubiquitin gene and expressed as ubiquitin-protein G fusion protein in an E. coli pET 22 expression vector (Novagen). The protein G sequence was in turn amplified from the ubiquitin-protein G fusion plasmid by using the primers 5′-GGTCTCAAGGTACGCCGGCGGTGACCACCT-3′ (SEQ ID NO:51) and 5′-AAGCTTATTATTCGGTCACGGTAAAGGTTT-3′ (SEQ ID NO:52) and inserted in pSUMO to generate pSUMO-protein G expression vector.
Construction of E. coli SUMO-β-galactosidase Expression Vector.
E. coli β-galctosidase was amplified using pfu (Stratagene) a preparation of genomic DNA from BL21(DE3) (Stratagene) as a template and the primers 5′-GGTCTCAAGGTATGACCATGATTACGGATTCACT-3′ (SEQ ID NO:53) and 5′-AAGCTTATTATTATTATTTTTGACACCAGACC-3′ (SEQ ID NO:54). The PCR products were purified and double digested with Bsa I and Hind III. These were then ligated into the vector pET24d6�HisSUMO, which had been similarly digested.
Construction of E. coli pSUMO-Liver X Receptor (LXR) Expression Vector:
The PCR products of the LXR from amino acid residue 189 to the end of the protein that spans the ligand binding domain was digested with BsaI and HindIII and ligated into the pSUMO vector, also digested with Bsa1 and HindIII.
Construction of E. coli pSUMO-MAPKAP2 Expression Vector:
The fragment of MAPKAP2, encoded in the plasmid pMON45641, was amplified by PCR and cloned into pET24d 6HisSUMO vector by designing PCR primers that flank the sequence shown
Construction of E. coli pSUMO-Tyrosine Kinase Expression Vector:
For the tyrosine kinase, both, the SUMO fusion and unfused expression vectors were designed. As described above the region of kinase was cloned by PCR flanked with BsaI and Hind III sites that were cloned in to similarly digested pSUMO.
Construction of E. coli pSUMO-β-Glucuronidase Expression Vector:
E. coli β-glucuronidase was the kind gift of Ben Glick, University of Chicago) and amplified with the primers 5′-GGTCTCAAGGTATGCAGATCTTCGTCAAGACGTT-3′ (SEQ ID NO:55) and 5′-AAGC TTATTATTGTTTGCCTCCCTGCTGCG-3′ (SEQ ID NO:56).
Construction of E. coli SUMO-Hydrolase Expression Vector:
C-terminal His-tagged SUMO hydrolase/protease Ulp (403�621)p (21) (27) was expressed from pET24d in Rosetta (DE3) pLysS (Novagen). The recombinant protein was purified using Ni-NTA agarose (Qiagen) and buffer exchanged into 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 5 mM β-mercaptoethanol using a PD-10 column (AP Biotech). About 2 ug of the pure protein was analyzed on gels and data shown in
Construction of E. coli UBL-GFP Fusion Vectors.
DNA sequences encoding ubiquitin (Ub), SUMO, Urm1, Hub1, Rub1, Apg8, and Apg12 were PCR-amplified using Deep-Vent polymerase (NEB) and yeast strain DNA to generate a template. Full-length human ISG15 cDNA was a kind gift of Dr. A. Haas, Medical College of Wisconsin, Milwaukee. A unique NcoI site followed by 6His sequence was introduced by PCR at the 5′-end of each Ubl cDNA. Primer sequence at the 3′-end included unique Esp3I and HindIII sites. PCR products were digested with NcoI/HindIII and inserted into respective sites of pET24d vector (Novagen) as described above. Full length GFP sequence (Clontech Cat # 60610-1) flanked by Esp3I and HindIII sites, respectively, was PCR-amplified and cloned into pCR4-TOPO-TA vector (Invitrogen). Esp3I/HindIII digested GFP-encoding gene was inserted into respective sites of pET24d-UBLl plasmids, creating final UBL-GFP expression vectors for E. coli. In toto, there were nine plasmid constructs coding for the following structures: 6His-Ubl-GFP. All plasmids were sequenced to confirm the expected structure.
Design and Construction of Yeast UBL-Fusion Vectors:
Saccharomyces cerevisiae has been used as a eukaryotic model for all the experiments involving yeast. All of the expression vectors for these studies were designed on multicopy yeast vectors that contain tryptophan or leucine as a selectable marker and 2μ as an origin of replication (22). Proteins were expressed as unfused products or as ubiquitin, SUMO or other UBL fusion proteins.
To demonstrate that UBLs increase the level of secretion of the protein to the media, in addition to enhancing the level of expression, expression vectors were constructed with and without ubiquitin. We have also compared ubiquitin fusion and SUMO fusion using GFP as a model protein (see
Construction of SUMO-N-GFP Yeast Expression Vector:
To determine what variety of N-terminal variant amino acids at the junction of SUMO and GFP can be cleaved in yeast we designed SUMO-GFP vectors in which all 20 amino acid residues were encoded at the N-terminus of GFP. Essentially all 20 SUMO-X-GFP vectors designed for E. coli expression were digested with Bsa I-Hind III, and the inserts were purified. The 20 inserts were cloned in Yep12 that was slightly modified. Specifically, YeEpSW was generated by digesting Yep12 with Bam HI and SacI. The CUP1 promoter region was recovered from the fragment by PCR. A polylinker was created at the 3′ end of CUP1 with a variety of restriction sites including NcoI and Xho1. All 20 SUMO-GFPs (N end variants) were digested with NcoI-XhoI enzymes and cloned directly YepSW. The resultant vector YepSW-SUMO-eGFP utilizes tryptophan selection and expresses SUMO-GFP proteins under the control of the copper promoter. All vectors were sequenced to ensure correct codons at the junction of SUMO and GFP.
Construction of UBL-GFP Fusion Yeast Expression Vectors:
Construction of the UBL-GFP fusion vectors for E. coli has been described above. In order to make UBL yeast expression vector NcoI/XhoI fragments carrying GFP alone and all the Ubl-GFP fusions were inserted into respective sites of pYEp SW (see above) that was similarly digested with NcoI/XhoI. Insertion of UBL-GFP cassette in Yep SW (See FIGS. 39 and 40A�40F), allows copper inducible expression of Ubl-GFP fusions in yeast system.
Design and Construction of Recombinant Baculovirus for SUMO and Ubiquitin GFP Fusion Expression:
To demonstrate that attachment of SUMO or ubiquitin to GFP increases its expression and enhances secretion into the media, several GFP fusion vectors were designed with different configurations of gp67 secretory signals. The basic GFP vector for expression is essentially based on E. coli vectors described above. Derivatives of this vector representing each candidate gene have been constructed by designing PCR primers. The construction of GFP plasmid transfer vectors for baculovirus is described. To help appreciate the rationale for the secretory signal in the context of GFP-fusion, see the diagrammatic representation shown in
(i) pFastbacE. A synthetic oligonucleotide containing the Esp3I site was inserted between BamHI and EcoRI cloning site of the transfer vector pFastbac1, which had been modified by removing Esp3I site from Gmr region. (ii) pFastbacG. The signal sequence of the gp67 gene derived from pACSecG2T was isolated by PCR using 2 primers (f-gp67 and r-gp67), digested with Bg1II and EcoRI in the next step, and then inserted between BamHI and EcoRI cloning sites of the transfer vector pFastbacE. (iii) pFastbacS. A full-length SUMO gene derived from pET SUMO was generated by PCR using 2 primers (f-bacsmt and r-bacsmt), digested with BsaI and EcoRI in the next step, and then inserted between BamHI and EcoRI cloning sites of the transfer vector pFastbacE. (iv) pFastbacG/S. The signal sequence of the gp67 gene in the pACSecG2T vector was generated by PCR using 2 primers (f-fusgp67 and r-fusgp67), and inserted between BamHI and EcoRI cloning sites of the transfer vector pFastbacE to create a new pFastbacG, which was used for fusion with SUMO afterward. A full-length SUMO gene derived from pET SUMO as described above (iii) was digested with BsaI and SacI and inserted between Esp3I and SacI cloning sites of the new transfer vector pFastbacG. (v) pFastbacS/G. A full-length SUMO gene derived from pET SUMO was generated by PCR using 2 primers (f-fussmt3 and r-fusgp67) and inserted between BamHI and EcoRI cloning sites of the transfer vector pFastbacE to create the new pFastbacS, used for fusion with gp67 afterward. The signal sequence of the gp67 gene derived from pACSecG2T as described above (ii) was digested with BsaI and SacI, and then inserted between the Esp3I and SacI cloning sites of the new transfer vector pFastbacS.
Preparation of Baculovirus Stocks and Cell Growth.
Transfer vector constructs based on the pFastbac 1 shuttle plasmid (Invitrogen, Inc.) were transposed in DH10Bac E. coli competent cells to transfer the respective e-GFP fusion sequences into recombinant virus DNA by site-specific integration. After alkaline lysis of transformed (white colonies) of E. coli cells, which contain recombinant virus (bacmid) DNA, and extraction of the recombinant bacmid DNA, the bacmid DNA was used to transfect Spodoptera frugiperda (Sf9) insect cells, in which virus replication occurs. The virus was then amplified to produce passage 2 (for long-term storage) and passage 3 virus (for working) stocks by infection of fresh Sf9 cell cultures and used directly to infect cells for fusion protein expression. Virus infectivity (pfu/ml) was determined by titration in Sf9 cells using the BacPAK� Rapid Titer Kit (BD Sciences Clontech, Inc.). A 50 ml culture of Hi-Five cells at concentration of 1�106 cells/ml, was infected with recombinant virus at MOI=5 in Express Five media (serum free media). The cells were grown in 100 ml spinner flask at 27� C. Every 24 hours, cell viability was determined by trypan blue and cell counting. 5 ml of the suspension culture was removed at 24 hour intervals, centrifuged at 500�g at 4� C. in 10 minutes. The supernatant was transferred into a fresh tube to monitor any protein that may have been secreted into the media (see below).
Analysis of Proteins from Insect Cell Compartments:
Cell pellets (from above step) were gently washed in 1 ml PBS and recentrifuged at 500�g at 4� C. for 10 minutes. All supernatant and pellets are stored at −80� C. The presence of recombinant protein in cells and media was ascertained by SDS-PAGE and Western blotting of supernatant and cell pellets. The total intracellular protein was extracted by M-PER extraction buffer (Pierce), a neutral buffer for protein extraction. The cell pellet was mixed with rapid pipetting and incubated for 1 hour on an orbital shaker. The suspension was centrifuged at 500�g at 4� C. for 10 minutes to remove debris. The supernatant contained extracted cellular proteins that were either analyzed by PAGE or stored at −80� C. To analyze the proteins present in the media, the following procedure was adopted. Trichloroacetic acid was added to 5 ml media to a final concentration of 20%. The suspension was mixed well and left on ice for three hours, and then centrifuged 500�g at 4� C. for 10 minutes. The white pellet was washed with 80% ethyl alcohol twice, and then dried. The pellet was suspended in 1 ml of M-PER buffer for PAGE to compare the distribution of control (unfused) and SUMO-fused proteins inside and outside the cell.
Methods for Analysis of Yeast Expressed Fusion Proteins:
Yeast cultures were grown in synthetic or rich media. Standard yeast and E. coli media were prepared as described (31). The yeast strain Y4727: Mata his3-Δ200 leu2-Δ0 lys2-Δ0 met5-Δ0 trp1-Δ63 ura3-Δ0 was used as a host (gift from Dr. Jeff Boeke) or BJ 1991. Yeast transformation was performed according to published procedures (8). Yeast transformants with autonomously replicating plasmids were maintained in yeast selective media. The E. coli β-Galactosidase and β-Glucuronidase proteins were expressed under the regulation of either the alcohol dehydrogenase (ADH), or Glyceraldehyde-Phosphate-Dehydrogenase (GPD) promoter or copper metallothioneine (CUP1) promoter in 2 μm multicopy plasmids with the LEU2 selective marker.
Yeast cells were transformed with appropriate expression vectors, and single colonies were grown in synthetic media minus the selectable marker. For each protein, at least two single colonies were independently analyzed for protein expression. Cells were grown in 5 ml culture overnight and, in the morning, the culture was diluted to an O.D. at 600 nm of 0.5. If the gene was under the control of copper inducible promoter, copper sulfate was added to 100 μM and the culture was allowed to grow for at least three hours. Cells were pelleted at 2000�g for 5 minutes, washed with 10 mM Tris-EDTA buffer pH 7.5. If enzymatic assays were performed, cells were disrupted in assay buffer with glass beads, 2� times the volume of the pellet. Cells were centrifuged and the supernatant was recovered for enzymatic or protein analysis. Alternatively, if the level and the type of protein was analyzed by SDS-PAGE, cell pellet was suspended in SDS-PAGE buffer and boiled for 5 mins. The suspension was centrifuged, and 10�20 ul aliquots were run on 12% SDS-PAGE.
Measurement of β-GUS Activity from Yeast:
β-Glucuronidase (GUS) is a 65 kDa protein that is a useful marker for protein trafficking. We have used GUS to determine the role of N-terminal ubiquitin on secretion of GUS in yeast. Yeast cells were transformed with various GUS vectors, grown overnight in selective liquid media at 30� C., and diluted in the liquid selective media to 0.1 OD600 (OD culture). Yeast cells were incubated in the presence of inducer in shaker at 30� C. After 4 hours of incubation, 100 μl of 2� �Z� Sarcosine-ONPG buffer (120 mM Na2HPO4, 80 mM NaH2PO4, 20 mM KCl, 2 mM MgSO4, 100 mM β-mercaptoethanol, pH 7.0, 0.4% lauroyl sarcosine) was added. (The 2� �Z� Sarcosine-buffer is freshly prepared or stored at −20� C. prior use.) We used a fluorometric assay with 4-methylumbelliferyl β-D-glucuronide as the substrate for β-GUS assay. After incubation at 37� C. for 1 hour (t incubation), the reaction was stopped by adding 100 μl of quenching solution, 0.5 M Na2CO3. The GUS activity was determined by reading the plates in a fluorometric plate reader. For calorimetric reactions, relative activity was calculated as following: (1000�OD reaction)/(t incubation�OD culture).
E. coli Growth, Compartmentalization and Protein Expression.
Protein expression studies were carried out in the Rosetta bacterial strain (Novagen). This strain is derived from the lambda DE3 lysogen strain and carries a chromosomal copy of the IPTG inducible T7 RNA polymerase along with tRNAs on a pACYC based plasmid. Cultures were grown in LB as well as minimal media and at growth temperatures of 37� C. and 20� C. with 100 ug/mL ampicillin and 30 ug/mL chloramphenicol. The culture was diluted 50 fold and grown to mid log (OD at 600 nm=0.5�0.7), at which time the culture was induced with 1 mM IPTG. Induction was allowed to proceed for 4�5 hrs. Upon completion of induction, cells were centrifuged and resuspended in a buffer containing 20% sucrose. To analyze protein induction in total cells, SDS-PAGE buffer was added and the protein was analyzed following SDS-PAGE and staining with Coomassie blue.
Separation of Soluble and Insoluble Fractions.
E. coli were harvested by mild centrifugation and washed once with PBS buffer. Cells were resuspended in 4 ml of PBS and ruptured by several pulses of sonication. Unbroken cells were removed by mild centrifugation (5 min at 1500�g) and supernatants were sonicated again to ensure complete cell lysis. An aliquot (5 μl) was mixed with 2% SDS to ensure that no viscosity is detected owing to lysis of unbroken cells. After ensuring that no unbroken cells remained in the lysate, insoluble material consisting of cell walls, inclusion bodies and membrane fragments was sedimented by centrifugation (18,000�g for 10 min). The supernatant was considered �Soluble fraction�.
The pellets were washed from any remaining soluble proteins, lipids and peptidoglycan as follows. Pellets were resuspended in 600 μl of PBS and to the suspensions 600 μl of solution containing 3 M urea and 1% Tri ton X100 was added. The suspension was briefly vortexed and insoluble material was collected by centrifugation as above. The PBS/Urea/Triton wash was repeated two more times to ensure complete removal of soluble proteins. The washed pellets, designated as �insoluble fraction,� consisted primarily of inclusion bodies formed by over expressed proteins. Approximately 10 μg of protein from each fraction was resolved on 12% SDS-PAGE minigels and stained with Coomassie Brilliant Blue.
Fluorescence (GFP Activity) Assessment.
GFP fluorescence was measured in soluble fractions (approx. 0.1 mg of soluble protein in a final volume of 40 μl) using Fluoroscan Accent FL fluorometer (LabSystems) with Excitation 485 nm/Emission 510 nm filter set with the exposure set to 40 sec. The data are presented in Arbitrary Units (AU).
Western Blotting.
Twenty μg of total yeast protein per lane were resolved on 12% SDS-PAGE minigel and electro-blotted to nitrocellulose membranes by standard methods. Membranes were blocked with 5% milk in TTBS buffer and incubated with rabbit anti-GFP antibodies (Clontech, cat no. 8367) at 1:100 dilution overnight at 4� C. Secondary HRP-conjugated antibodies were from Amersham. Identical gels were run in parallel and stained with Coomassie to ensure equal loading of the samples.
The various 6HisxSUMO-GFP (16) fusions were expressed in Rosetta (DE3) pLysS (Novagen) using the procedures recommended by the manufacturer. Expression levels in the absence and presence of the fusion proteins was compared by SDS-PAGE analysis. The recombinant proteins were purified using Ni-NTA agarose; (Qiagen) using procedures recommended by the manufacturer.
Cleavage of Proteins
For studies in E. coli, an organism that does not possess SUMO or ubiquitin cleaving enzymes, each cleavage reaction contained 100 ul of purified fusion protein, 99 ul of the buffer 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol, and 1 ul of enzyme. The reactions were incubated for 3 hours at 30� C., and stopped by addition of 6� Laemmli SDS-page loading buffer followed by boiling at 95� C. for 5 minutes. The products of the cleavage reaction were analyzed by SDS-PAGE.
The following examples are provided to illustrate various embodiments of the present invention. They are not intended to limit the invention in any way.
The design and construction of all the UBL E. coli expression vectors has been described above. The DNA sequences, accession numbers of the UBL-GFP fusion proteins, and translation frames are shown
In addition, it is shown that ISG15 plays a role in immune response (24). Thus presentation of ISG15 as a fusion protein is a viable tool for novel vaccine candidates. Similarly, Apg8 and Apg12 translocate protein to compartments in the cell for autophagy (30).
Similar experiments were performed with all the UBL-GFP fusion proteins, but the induction was performed at 26� C. overnight. The data shown in
To gain more insight into the role of UBLs in enhancement of expression and solubility, we have tested the SUMO-fusion systems with other proteins as well. Serine threonine kinases, tyrosine kinase and human nuclear receptor have proven difficult to express in E. coli. Researchers have opted to use tissue culture systems to express soluble kinases of receptors.
Human nuclear receptor proteins, such as steroid receptors, contain ligand-binding domains. These proteins have proven hard to express in soluble form in E. coli. We have used human liver X receptor (LXR) ligand binding domain to demonstrate that SUMO fusion promotes solubility of the protein in E. coli. The ligand-binding domain of LXR was expressed as SUMO fusion in Rosetta plysS cell at 20� C. or 37� C. and the pattern of soluble and insoluble protein was analyzed.
Overall, these results show that in bacteria, fusion of UBLs to GFP increases the level of expression from 2�40 fold. Some of the UBLs such as Ub, SUMO, Urm1, Hub1, and ISG15 solublize otherwise insoluble proteins. In particular, SUMO has been demonstrated to increase solubility of kinases and LXR α under controlled temperature induction from 50�95% of the total expressed protein.
Fusions of C-Terminal UBLs to the N-Terminus of GFPs are Cleaved in Yeast
To further assess the utility of UBL fusion in eukaryotic cells we expressed all of the UBL-GFP fusions previously described in
Generation of New Amino Termini:
The identity of the N-terminus of a protein has been proposed to control its half-life (the N-end Rule) (35). Many important biopharmaceuticals such as growth factors, chemokines, and other cellular proteins, require desired N-termini for therapeutic activity. It has not been possible to generate desired N-termini, as nature initiates translation from methionine, but the SUMO system offers a novel way to accomplish this.
To demonstrate that all N-termini of GFP in SUMO-GFP fusions were efficiently cleaved when expressed in yeast, a comprehensive study of SUMO-GFP with 20 N-termini was carried out. Multi-copy yeast expression plasmids were designed as described above. Plasmids were transformed in yeast strain BJ 1991, four single colonies were selected, and the levels and cleavage patterns of two of the strains were analyzed by SDS-PAGE and western blotting. Data from Western blots of a single colony is presented in
Previous studies have shown that attachment of ubiquitin to the N-termini of proteins in yeast enhances expression, and protein fusions containing all amino acid at the N-terminal residue, except proline, are efficiently cleaved in yeast (2, 10, 34). However, these technologies have several drawbacks. Firstly, none of the deubiquitinating enzymes (DUBs) have been shown to efficiently cleave ubiquitin fusion proteins of varying sizes and structures (3,1), despite the fact that they were discovered more than 15 years ago (35, 19, 3). Secondly, and perhaps more importantly, ubiquitin predominantly functions as a signal for proteolysis (14). Therefore, for physiological reasons and for the lack of robust cleavage of artificial ubiquitin-fusions by DUBs, the ubiquitin gene fusion system has not been successfully developed for commercial applications. We have observed that the SUMO system appears to perform in a manner that is remarkably superior to that of ubiquitin, as SUMO and other UBL fusions enhance protein expression and solubility in prokaryotes. In addition, many of the UBLs increase expression of GFP, following the cleavage of UBL in yeast. Unlike the ubiquitin-fusion system, which may direct the protein to the ubiquitin proteosome pathway, the current cleavage of fusion-protein in yeast is the result of C-terminal fusion with SUMO, and proteins generated with novel N-termini are not subject to degradation by the ubiquitin-proteosome pathway. This is one of the reasons that large amount of GFP has accumulated in yeast after cleavage of the SUMO fusion (see
N-Terminal Attachment of Ubiquitin Promotes Protein Secretion:
To date, a role for ubiquitin in the secretion of proteins has not been determined. We have assessed whether N-terminal fusion of ubiquitin to a protein promotes its secretion in yeast. Several yeast expression vectors that express E. coli β-glucoronidase (GUS) were designed. All of the yeast GUS expression vectors described in Table 2 are engineered under the control of the strong glycolytic GPD promoter that expresses constitutively. Some of the constructs were also expressed under the control of a copper regulated metallothionein promoter (CUP1) as well. CUP1 promoter driven synthesis of the SUMO-GUS constructs was induced by addition of 100 μM copper sulfate and incubation of 3 hours. To determine the level of GUS from media, cells were harvested by centrifugation at 2000�g for 10 mins. Supernatant was collected and equal amounts of aliquots were assayed for enzymatic activity or western blot analysis as described above. For the comparative study, all strains were treated identically and grown at the same time to equal O.D, and the assays were performed at the same time. To examine intracellular enzymatic activity, the cells were harvested by centrifugation and washed with Tris EDTA buffer, pH 7.5. The cell pellets were suspended in sarcosine buffer and ruptured with glass beads at 4� C., three times by vigorously vortexing. Supernatant was collected for assay of the enzymatic activity. The amount of protein secretion was determined by estimating relative activity of the enzyme in the media. The data is shown in Table 2.
The role of SUMO in enhanced expression and secretion of proteins in cultured cells has also been studied in insect cells. Baculovirus vectors expressing SUMO-GFP constructs and appropriate controls have been described above. See
Similar experiments were performed with cells 48 hours post infection. The data in
We have also discovered that SUMO-Pro-GFP fusion was not cleaved by endogenous SUMO hydrolases in insect cells (
Further confirmation of these observations was obtained by fluorescence imaging of the cells expressing GFP fusion proteins.
SUMO Protease ULP1 Cleaves a Variety of SUMO-Fusion Proteins:
Properties and Applications in Protein and Peptide Expression and Purification
Yeast cells contain two SUMO proteases, Ulp1 and Ulp2, which cleave sumoylated proteins in the cell. At least eight SUMO hydrolases have been identified in mammalian systems. The yeast SUMO hydrolase Ulp1 catalyzes two reactions. It processes full length SUMO into its mature form and it also de-conjugates SUMO from side chain lysines of target proteins. Examples I and II establish our findings that attachment of SUMO to the N-terminus of under-expressed proteins dramatically enhances their expression in E. coli, yeast and insect cells. To broaden the application of SUMO fusion technology as a tool for expression of proteins and peptides of different sizes and structures, the ability of Ulp1 to cleave a variety of proteins and peptides has been examined. Purified recombinant SUMO-GFPs were efficiently cleaved when any amino acid except Proline is present in the +1 position of the cleavage site. Similar properties of SUMO hydrolase Ulp1 were observed when Sumo-tyrosine kinase, Sumo-protein G, Sumo-β-GUS, and SUMO MAPKAP2 kinase were used as substrates. The in vitro activity of the enzyme showed that it was active under broad ranges of pH, temperature, and salt and imidazole concentration. These findings suggest that the Ulp1 is much more robust in cleavage of the SUMO-fusion proteins as compared to its counterpart, ubiquitin-fusion hydrolase. Broad specificity and highly efficient cleavage properties of the Ulp1 indicate that SUMO-fusion technology can be used as a universal tag to purify a variety of proteins and peptides, which are readily cleaved to render highly pure proteins.
The following materials and methods are provided to facilitate the practice of Example III.
Affinity Purification and Cleavage of SUMO Fusion Proteins with SUMO Hydrolase.
The following table lists the solutions required for the affinity purification and cleavage procedures:
After cleavage, protein can be stored at 4� C., or subjected to purification.
The expression and purification of carboxy terminus of Ulp1p is described above.
The various His6smt3XeGFP fusions were expressed in Rosetta (DE3) pLysS (Novagen). The recombinant proteins were purified using Ni-NTA agarose (Qiagen). The comparative in vitro cleavage reactions were carried out by first normalizing the amount of the various fusions in each reaction. This was done by measuring the fluorescence properties of the purified fusion proteins using the fluorimeter Fluoriskan II (Lab Systems) and then diluting the more concentrated samples with the Ni-NTA agarose elution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl 300 mM Imidazole and 5 mM beta-mercaptoethanol), such that their fluorescence values equaled that of the lowest yielder. Each cleavage reaction contained 100 ul of protein, 99 ul of the buffer 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 5 mM beta-mercaptoethanol and 1 ul of enzyme. The reactions were incubated for 3 hours at 30� C. after which they were stopped by addition of 6� Laemmli SDS-page loading buffer followed by boiling at 95� C. for 5 minutes. The products of the cleavage reaction were analyzed by SDS-PAGE.
Proline cleavage experiments were carried out in a fashion similar to those described above. The purified His6smt3PeGFP was buffer exchanged into 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 5 mM beta-mercaptoethanol using a PD-10 column. A 10 fold increase in the amount of Ulp1 were added to each reaction. Digestions were incubated for 3 hours at 30� C. All reactions were stopped by addition of Laemmli loading buffer and analyzed by SDS-page.
Conjugation of ubiquitin and SUMO to its target proteins is a highly regulated and dynamic process. Several deubiquitinating enzymes (DUBs) have been identified in yeast and other eukaryotic cells (1). Yeast genetics studies show that many of these enzymes are not essential suggesting that an overlapping function is performed by most of these enzymes. DUBs have been most extensively studied and shown to cleave linear ubiquitin fusions as well isopepetide bonds (3, 35). Much less is known about the enzymes that remove SUMO from isopeptide bonds or artificial SUMO-fusion proteins. Hochstrasser and Li have shown that Ulp1 and Ulp2 remove Smt3 and SUMO 1 from proteins and play a role in progression through the G2/M phase and recovery of cells from checkpoint arrest, respectively (20, 21). Ulp1 and Ulp2 cleave C-terminus of SUMO (−GGATY; SEQ ID NO: 59) to mature form (−GG) and de-conjugate Smt3 from the side chains of lysines (20, 21). The sequence similarity of two enzymes is restricted to a 200-amino acid sequence called ULP that contains the catalytically active region. The three-dimensional structure of the ULP domain from Ulp1 has been determined in a complex form with SUMO (Smt3) precursor (27). These studies show that conserved surfaces of SUMO determine the processing and de-conjugation of SUMO. Database searches of the human genome and recent findings suggest that there are at least 7 human ULPs with the size ranging from 238 to 1112 amino acid residues (18, 33, 39). It is intriguing to note that SUMO Ulps are not related to DUBs, suggesting that SUMO Ulps evolved separately from DUBs. The findings that ULP structure is distantly related to adenovirus processing protease, intracellular pathogen Chlammydia trachomatis and other proposed bacterial cystiene protease core domains suggest that this sequence evolved in prokaryotes (20, 21). Detailed properties of the SUMO proteases are provided in described in Table 3.
The effects of various additives/conditions and temperature upon the in vitro cleavage reaction were determined as follows: His6smt3MeGFP was expressed from pET24d in Rosetta (DE3) pLysS (Novagen). The recombinant protein was purified as before using Ni-NTA agarose (Qiagen) and then buffer exchanged into 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 5 mM β-mercaptoethanol using a PD-10 column (AP Biotech). Cleavage reactions were performed with 100 ug of the purified protein, 0.5 ul of enzyme, the appropriate amount of a stock solution of additive to generate the final concentrations listed in Table 4, plus the exchange buffer up to a final volume of 200 ul. Reactions were incubated for 1 hour at 37� C. except for those at 4� C. were incubated for 3 hours. The data in
The SUMO hydrolase also functions under broad pH range.
As discussed above, for broad utility of the system it is important that the enzyme be able to cleave fusion proteins of different sizes and structures in vitro.
Among dozens of proteins expressed as SUMO fusions in our lab, only one, β-GUS, proved partially resistant to cleavage by the hydrolase. Configurations of artificial SUMO fusion are bound to occur wherein the structure of the protein will hinder the ability of the enzyme to recognize and bind the cleavage site of the fusion protein. This problem has been solved by adding small concentrations of urea, which does not inhibit the hydrolase, but results in cleavage the fusion that was previously resistant.
We have discovered that, due to the rapid folding properties of SUMO, the fused protein can also be rapidly re-natured after treatment of the crude protein mix with chaotropic agents such as guanidinium hydrochloride or urea. We have developed a simple and rapid procedure to purify SUMO-fused proteins that are expressed in prokaryotes and eukaryotes. This method was tested with SUMO-protein G fusion expressed in E. coli. Cells expressing 6�His-SUMO-G protein fusion were harvested and frozen until required for protein purification. Three times the weight per volume lysis buffer (6 M Guanidinium Chloride, 20 mM Tris-HCl, 150 mM NaCl, pH 8.0) was added to the cell pellet rapidly lyse the cells. The supernatant was loaded onto a pre-equilibrated column containing Ni-NTA agarose (Qiagen), the flow through was collected for analysis. The column was then washed, first with 2 column volumes (CV) of Lysis buffer, followed by 3 CV of wash buffer (20 mM Tris-HCl, 150 mM NaCl 15 mM Imidazole pH 8.0). The fusion protein was then eluted using 2 CV of elution buffer (20 mM Tris-HCl, 150 mM NaCl 300 mM Imidazole pH 8.0). The purified product is present in a native buffer that allows for cleavage and release of the peptide from the Sumo fusion using Ulp1. See
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.
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OP Senior Member
Thanks Meter: 364
380 posts
Join Date:Joined: Oct 2011
I will release XAP here as soon as.
I am happy UnZip MortScript command works well on WP7. Then we are able to simply unzip all ZIP and XAP archives downloaded from XDA by internet explorer on unlocked devices, PC using is unnecessary now. I can, for example, add simply plugin to Phone Commander application for archives unzipping from actual selection to inactive panel directory (like Total Commander internal unzipper).
Last edited by Martin7Pro; 29th January 2014 at 08:39 PM.


text:
|Half Baths
|Property type
|Minimum Stay
|3 - 7 nights
This beautiful custom built home is located one block from the beach at 3605 Cameron Boulevard and is 15 minutes from historic downtown Charleston and many championship golf courses.
This six bedroom house is tastefully decorated with beautiful furnishings, a large den with a entertainment system, flat screen televisions in great room and bedrooms, fantastic gourmet kitchen and 2 master suites (one with a jacuzzi tub).
Come straight from the beach and shower in the privacy of an outdoor shower. Take the elevator to the greatroom and walk out on the porch and sit in one of the rocking chairs and feel the ocean breeze. For a spectacular look at the great Atlantic Ocean take a walk up the spiral staircase for breath taking views.
This home has a Non-Smoking and has a No Pet policy.
Keywords: Beach House
We are family owned sharing the same experience that we grew up as kids spending time on the Isle of Palms and enjoying the beach.
Brian Johnson purchased this house in 2003
We have put every item that we enjoy when going on vacation. From flat screen televisions in every bedroom to the ping pong table to have a family tournament during your stay. The rocking chairs on the porches to sit outside and enjoy the view and beach breeze.
Locally Owned by the Johnson Family
We thoroughly enjoyed your beautiful home. The porches and views were spectacular. The furnishing and decorations made you feel as I was home. We look forward to returning next summer.
Very attentive owner. House was perfect for our three family needs. Ping pong table was a plus and kept the young adults entertained. Close enough to Charleston to go in almost every day and a very short walk to beach.
This spacious beach house met our family's needs, perfectly! Though we're local, most of our guests came from out of town and visiting the beach is always a treat! The elevator and wide hallways are a necessity with older guests (to 90's!), giving them the independence they need. Open living/dining layout made mealtime/visiting fun and convenient! The kitchen is well equipped with necessary appliances and everything you'd need for meals. The large pantry worked well, as we brought in lots of food and supplies, simplifying our stay. The bedrooms and baths are amply-sized and the layout assured everyone of privacy. We experienced an unseasonal cold snap and weren't able to enjoy the porches as we'd hoped (that's OK, there's always next time!). The pictures represented the property accurately and we were very pleased to find everything in good, clean condition. The city is watchful with overnight parking and having the ability to park underneath was certainly a plus! We appreciate the owners providing plenty of ice when they realized the ice maker wasn't working. They were very attentive to our needs and we appreciate their personal interest!
This is a great house. The arrangement of bathrooms and bedrooms worked well for our extended family. The kitchen was well equipped with everything we needed. The ice maker kept pace. The owner was very prompt with our questions upon arrival. Quick return on phone calls. The proximity to Charleston was well appreciated. The beach is a block away, a do-able walk.
A great house for 2 families to share. We had 4 adults and 7 children (21+ years old). Kitchen was stocked well, the rooms were comfortable, and the decks were great. The walk to the beach was easy. The 21+ year old kids enjoyed the upper deck at night. They did not disturb the adults when they went to bed. All in all, the house was great. My only complaint would be that the Master Bedrooms were located below the great room and kitchen. For guests that went to bed early, you had to be careful when you walked around upstairs.
We stayed in this beautiful home for the Christmas holidays with four of our children and five grandchildren. There was plenty of space for everyone, and plenty of attractions to keep us busy for the week. Beach, fishing (we caught a shark), golfing, ice skating, sightseeing. We drove through the Holiday of Lights and roasted marshmallows and made s'mores. We went and toured Magnolia Plantation and gardens. The kitchen was well stocked for cooking and serving. The elevator made bringing luggage and groceries into the house easy. It wasn't the right time of year to enjoy the upper deck of the house, but I'm sure it's great during the summer months. It's a very easy walk to the beach, even for the little ones. We drove into Charleston several times and found it to be a pretty easy drive. We highly recommend this property.
Our Senior Adults enjoyed spending an extended weekend in your home. The house was most impressive. The kitchen had all of the items needed for preparing our meals. The floor plan made it easy to visit with each other, yet we each had our own space as well. The elevator made it easy to get our kitchen supplies up to the second floor. We had a great weekend and made lots of memories!
Other Activities: basketball court, horseshoes, boating, shopping
|Rate Period
|Weekend Night
Mar 1 2015 - Apr 17 2015
3 night minimum stay
Apr 18 2015 - Jun 5 2015
3 night minimum stay
Jun 6 2015 - Sep 7 2015
7 night minimum stay
Fall 2015
Sep 8 2015 - Oct 2 2015
3 night minimum stay
Winter 2015
Oct 3 2015 - Mar 4 2016
3 night minimum stay
|Refundable damage deposit
|Tax Rate
Payment is usually accepted in the quoted currency (USD) unless the currency and the amount is specifically agreed in advance with the owner / advertiser.


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The girlfriend of Natasha McShane's attacker has been held on a $1 million bail after prosecutors said she was "just as culpable and as guilty for every whack of the bat."
Moments before they attacked Natasha McShane and Stacy Jurich, Marcy Cruz told Heriberto Viramontes to "look at all those drunk a-------s coming out of the bar."
Assistant Cook County State's Attorney Erin Antonietti said Cruz pointed out the potential victims to Viramontes after he told her he needed "quick money for drugs."
Cruz told police that she "smoked a blunt" of marijuana before the attack took place.
Moments late Viramontes jumped out of their van and brutally beat the students around the head with an aluminum baseball bat.
Cruz took perfume and make up from the girls' purses, and she was filmed using the credit cards of the victims at a nearby gas station.
Antonietti said the students "could not have been attacked" without Cruz's assistance and she said Cruz was "as culpable and as guilty for every whack of the bat as defendant Viramontes."
The parents of Natasha McShane have flown to Chicago to be at her bedside.
They are "devastated and heartbroken" by the whole ordeal.
Doctors are unsure about McShane's future, if she awakes from the coma she is unlikely to walk or talk again.
In a statement released by Northwestern Memorial Hospital, McShane's parents said she "is everything a parent could ever wish for in a daughter."
They thanked "all of the people who have reached out . . . with offers of help and prayer."
They are "greatly comforted by the overwhelming support from friends and strangers alike -- a support that is growing daily as people read how critical her condition is."
The McShanes asked the public to "keep Natasha in your thoughts and prayers during these critical coming weeks".
The family also thanked the medical staff at Northwestern Memorial Hospital.
For those of you that may wish to help the family by contributing to McShane's medical bills, you can donate to the Natasha McShane Care Account at North Bank, 501 N. Clark St., Chicago, IL 60654.
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Jun 11, 2013; Eden Prairie, MN, USA; Minnesota Vikings quarterback Christian Ponder (7) passes in drills at the Minnesota Vikings Minicamp at Winter Park. Mandatory Credit: Bruce Kluckhohn-USA TODAY Sports
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Apollo wants you to enjoy time with your family at Cragun's! Win a 2 night stay at Cragun's on Gull...
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Need help with baking clay
To make a long story short, I had to have one of my dogs' put to sleep and cremated. The cremation service sent me an un-baked clay medallion with my dog's name on it and his paw print. This was a total surprise to me! Anyway it came with instructions. It is un-baked and I do not know what type of clay it is, but it says to bake in conventional or toaster oven at 275 degrees on a GLASS baking dish. First of all I have never seen a glass dish for use with a toaster oven. I do have Pyrex but they will not fit into my toaster oven and my Pyrex pie plate is the only one almost flat enough to put in a conventional oven with the clay piece in the center. I was planning on using the toaster oven for the purpose of convenience, but the flat Pyrex glass pieces I have seen at Wal-Mart say not to use in toaster ovens. The clay instructions also state not to use metal.
Any suggestions for me?
Wow, can't believe no one has answered your harmless question! I hope by now you've figured out to handle this but in case you haven't, you can use a flat ceramic tile.
This is my first time checking out this forum BTW, I'm just learning polymer clay myself and am addicted already, can't believe the low traffic on here.


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Staff Reporter
KHAMMAM: Members of the All India Democratic Women's Association (AIDWA), Khammam district unit, on Tuesday took out a rally from the municipal office to the Collectorate demanding steps to streamline the public distribution system.
Denouncing the failure of the government to control the spiralling prices of essential commodities, they staged a demonstration in front of the Collector's office. A delegation of the AIDWA leaders allowed into the Collectorate complex to submit a memorandum to the district revenue officer.
Led by M. Jyothi and other members of the Mahila Sangham, the protesters raised slogans against the delay in issue of white ration cards. Genuine applicants have been frequenting to the Tahasildar offices for the past four years for the ration cards, but in vain in many cases. They said that rations shops had different schedules for supply of rice, sugar, dal and edible oils. The card-holders were fed up visiting the fair price shop five to six times a month.
They threatened to step up the agitation if the government failed to streamline the distribution of essential commodities. AIDWA leaders – M. Lakshmi and B. Sarala -- were among those who took part in the protest.


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Bellview Di Rosa
No availability found through our partners. Please contact the business directly or check some of our recommended alternatives.
TripAdvisor Reviews Bellview Di Rosa Taormina
Travel Blogs from Taormina
Storm of the century & centuries-old salt & stone
... homelike than a fabulous Swiss village or seaside Sicily. There's homework to be done, uniforms to wash, Christmas thank you notes to write. There's six months ahead of us -- to chart the rest of our course during this now NEW year of both the every day and the extra special.
Happy New Year, friends far and near. We are thankful for your place in our lives and look forward to time together to share more stories.
Greek Theatre
... the Romans picked it up and added their own influence. The Romans were able to built the arts because of the power of the empire. Only when citizens feel safe and provided for, can they begin to focus on things like dramatic performance. But because of the drastic power the empire possessed, the richness of culture grew with it. This was revolutionary for culture and society of its ...
Taormina to Palermo
Today we travelled from Taormina (left about 10.30am) to Palermo with Carmelo who was a great driver. He brought us safely to excelsior palace after about 3 hours. We had a bit of a walk around Palermo before heading out for dinner. It is sometimes an interesting experience with the ...
Today we had a fairly leisurely morning before heading out for lunch to a nice ristorante "Grandula" with a nice view. We then caught a taxi and checked out the harbour down the hill which we have been viewing all week. We also viewed the first Greek colony of Sicily founded in 734BC by ...
... dinner always punctuated by interesting translation details but dinner usually ends up being nice. Tonight we had a very cute old Italian waiter who told John when he ordered chips that chips were for kids and he needed to look after his liver. Nevertheless John ordered chips!! Maxine's stuffed zucchini flowers never arrived and our Italian waiter was so ...
Other places to stay in Taormina
ATAHOTEL CapotaorminaVia Nazionale 105, Taormina | 4 star hotelfrom $269
- Enchanteur et haut de gamme !
- Average
- Awesome hotel
Monte Tauro HotelVia Madonna delle Grazie 3, Taormina | 4 star hotelfrom $327
- Fantastic stay at the Monte Tauro
- Not up to scratch!
- Selective information.....
San Domenico Palace HotelPiazza San Domenico 5, Taormina | 5 star hotelfrom $401
- Gorgeous gardens, beautiful public rooms, disappointing bedrooms
- Wonderful and unique - like staying in a living museum
- Paradise
Belmond Grand Hotel TimeoVia Teatro Greco 59, Taormina | 5 star hotelfrom $849
- Stunning service, views and rooms
- An oasis at the end of a long vacation
- Grand Prices, Grand View but no Grand Hotel
Caparena Hotel189 Via Nazionale, Taormina | 4 star hotelfrom $182
Hotel Villa DiodoroBagnoli Croci 75, Taormina | 4 star hotelfrom $237
- Excellent location, staff rude and disapointing food
- Great Hotel, Not So Great Staff!
- Excellent hotel and excellent staff
Mazzaro Sea PalaceVia Nazionale 147, Taormina | 5 star hotelfrom $269
Villa BelvedereVia Bagnoli Croci, 79, Taormina | 3 star hotelfrom $245
- Villa Belvedere - rude, arrogant, inhospitable
- Lovely hotel with beautiful views
- Terrific situation
Grand Hotel San PietroVia Pirandello, 50, Taormina | 5 star hotelfrom $352
- Un vero 5 stelle lusso!
- Wunderbares San Pietro
- Superhotel in guter Lage
Grand Hotel MiramareVia Guardiola Vecchia, 27 , Taormina | 4 star hotelfrom $187
- Great Hotel
- Fab Holiday
- Fantastic hotel, town and island
Grand Hotel Atlantis BayVia Nazionale, 161, Taormina | 5 star hotelfrom $367
TAORMINA PARK HOTELVia Bagnoli Croce 92, Taormina | 4 star hotelfrom $166
- Fabulous break
- Food dreadful, but stunning views
- Bristol Park Hotel August 2003
Hotel Villa ChiaraVia Don Bosco 10, Taormina | 2 star hotelfrom $90
La Plage ResortVia Nazionale 107/A, Taormina | 5 star hotelfrom $306
President Hotel SplendidVia Dietro Cappuccini, 10, Taormina | 3 star hotelfrom $100
Mediterranee HotelVia Circonvallazione, 61, Taormina | 4 star hotelfrom $151
Parc Hotel Ariston & Palazzo Santa CaterinaVia Bagnoli Croce 1, Taormina | 4 star hotelfrom $193
- A great hotel
- Only ok; lots of steps
- Good View But...
Bay Palace HotelVia Nazionale 114, Taormina | 3 star hotelfrom $111
Sole CastelloVia Leonardo da Vinci 83, Taormina | 3 star hotelfrom $96
Le Grand SoleilVia Dietro Cappuccini, Taormina | 2 star hotelfrom $97
Excelsior Palace HotelVia Toselli 8, Taormina | 4 star hotelfrom $167
- Excellent location - good hotel
- Price is not in proportion to quality offered
- Super hotel and location
Sirius HotelVia Guardiola Vecchia, 34-37, Taormina | 4 star hotelfrom $195
- Fabulous small hotel with great service and views
- Lovely hotel;great location;teriffic views
- Breathtaking views
Bel Soggiorno HotelVia L Pirandello 60, Taormina | 3 star hotelfrom $99
- Fantastic Place to stay!
- A great stay at the Bel Soggiorno
- We would happily return to Bel Soggiorno
Villa Bianca ResortPiazzale Della Funivia, Taormina | 3 star hotelfrom $113
Hotel CoralloVia Madonna delle Grazie 9, Taormina | 4 star hotelfrom $86


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Barcelo Costa Vasca
No availability found through our partners. Please contact the business directly or check some of our recommended alternatives.
- Suites
- Minbar in room
- Free High-Speed Internet
- Breakfast Available
- Wireless internet connection in public areas
- Room service
Photos of Barcelo Costa Vasca
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TravelPod Member ReviewsBarcelo Costa Vasca San Sebastián - Donostia
Nice hotel. Breakfast is basic although noticed after breakfast that you can ask staff for cooked eggs. Lovely big bathroom, possibly meant for the disabled. A view of the road and hillside at the back of the hotel. Over 2km from any restaurants or shops but no 15 bus is right outside.
This review is the subjective opinion of a TravelPod member and not of TravelPod.com.
Whilst the proximity to attractions and rooms are fine, there are a few internal procedures that need looking into.
This review is the subjective opinion of a TravelPod member and not of TravelPod.com.
TripAdvisor Reviews Barcelo Costa Vasca San Sebastián - Donostia
Travel Blogs from San Sebastián - Donostia
San Sebastián - To Barcelona!
13/6/14 Friday. Santander to San sebastian - only about 200km Hooray. A sleep-in we board the bus at 9.30am after another wonderful full buffet breakfast in a beautiful room with windows overlooking a rose garden. Best bed of the whole tour. A bit foggy for the first hour and head into Basque Country travelling on the E70 freeway. Pass some very …
Living the life in San Sebastian
... at a cart to get a little paddle game for the beach to help keep Jon occupied and not bored. Best 5€ ever spent since we played ever time we went to the beach and we're bringing them home. By the time we hung out at the beach and darkened our tans even more, we were ready to shower and grab some of these famous tapas, or pintxos as they were called, for dinner. We found one that looked amazing and just stayed there for our choice ...
San Sebastian - A gastronomic paradise
... historic, exuding an old world feeling. It reminded me of the Palace Hotel in San Francisco. Fortunately I was able to use Starwood points to stay here, and lucky I booked early because it was booked solid.
San Sebastian is considered the culinary capital of Spain, depending on who you talk to. They are famous for their Pintxo bars in the Old Town. Pintxos (tx is pronouced like ch as in church) are similar tapas, and they ...
San Sebastian
... the walk much slower. We made it back to the Boulevard and stopped to eat again. Finished up the lunch items. Then at 9:15 we made our way over to Ain's area. Got a little lost, but then made our way as we usually do. Found parking and met up with Ain. Got settled in his place and then went out for dinner. We opted for Lebanese food. It was okay. I didn't like all of it, but ...
I´m moving to Basque Country. Goodbye friends...
... I am now. We have a bus back to Santander at 7:30pm and so we are just going to hang out until then. It is definitely too bad that we cant stay longer in this city. I would have loved to experience more of it and all that it has to offer. However, I did indeed have one of the greatest weekends of my life and don't regret a single minute of it. Next summer, I´m coming back... I miss you all very much (except when I´m dancing in the wee hours of the morning), -Alexandra ...


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Narrow your search
While it's a bit pricey, the sleek design of the Sharp i-Elegance DK-AP7N sets it apart from more mundane portable iPod speakers.
The 2015 GLA250, a new compact SUV from Mercedes-Benz, delivers on style and self-driving tech, while offering cabin appointments worthy of the brand.
The all-new Jaguar XE, on display at the Paris Motor Show, brings a mid-size sedan into the brand's lineup that will make an entry point for many people.
To celebrate not Star Wars day, but the birthday of Audrey Hepburn, Google takes one more step in making women more prominent in its doodles.
This feature-rich double oven is part of GE's high-end Profile line.
Form virtual pottery throwing to music making, these Android apps do wonders to get you out of your mental rut and try something new.
The Audiophiliac drops by the Audioarts showroom for an audition of these stunning speakers.
The 2014 Land Rover LR4 is the last of the chunky, square Land Rover left in North America. Let's see if it can still mix stylish elegance with off-road pedigree.
This animated short brilliantly taps Lego minifigs and CGI to re-create the ending of "The Time of The Doctor," where Matt Smith regenerates into Peter Capaldi. Now, about those pesky fish fingers.
For his concept of future Apple devices, one designer dreams very big. Check out his visions here.


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Seamar Hotel - Fortaleza
Rua Carlos Vasconcelos, 115 Praia de Iracema - Fortaleza1-877-662-6988
From families to romantic couples and solo travellers will love the hotel's location in Icarema Beach, the most beautiful beach in Fortaleza. The area offers a wide range of restaurants, drinking bars, and shopping choices.
On site, the hotel brasserie serves breakfast, lunch, and dinner, and limited-hour room service is available to guests. Additional hotel amenities include an outdoor swimming pool and poolside sun loungers/chaise longues, free wireless Internet access in the lobby, complimentary covered parking, and a restaurant.
Please note: minimum check-in age is 18.
We show TripAdvisor reviews for new Venere hotels that haven't been reviewed yet
Check Availability at Seamar Hotel
Breakfast at Seamar Hotel
A free buffet breakfast is available between 6:30 AM and 10:00 AM.
Facilities at Seamar Hotel
- Luggage room
- Multilingual staff
- City tour
- Front desk - 24 hour
- Lift/elevator
- Room service - limited hour
- Laundry room
- Internet/Email services
- Restaurant/Dining
- Outdoor swimming pool
- Free parking
Know before you go
- Parents traveling with a child under 18 must present the child's birth certificate and photo ID (passport for foreign visitors) upon check-in. If only one parent or legal guardian is traveling with the child, that parent or legal guardian must also present a notarized consent of travel signed by both parents. Visitors who plan to travel with children should consult with a Brazilian consulate office prior to travel for further guidance.
- Children 5 years old and younger stay free when occupying the parent or guardian's room, using existing bedding.
- Only registered guests are allowed in the guestrooms.
Check-in: after 1:00 PM.
Check-out: before noon.
PolicyExtra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
City tax and/or any other fees charged by the hotel
You'll be asked to pay the following charges at the hotel:
- Tourism fee: BRL 1.82 per person, per night
We have included all charges provided to us by the property. However, charges can vary, for example, based on length of stay or the room you book.
Room Details
Book your family-friendly accommodation at Seamar Hotel, offering 37 guest rooms with climate control. Televisions in each room offer entertainment at the end of a long day. Corporate travelers will benefit from free wireless high-speed Internet access as well as desks, safes and phones during their Beira Mar visit. Private bathrooms are outfitted with showers. Additional guest comforts include minibars, air conditioning and blackout drapes/curtains.
Accommodation Services
- Telephone
- Air conditioning
- Mini bar
- Safe
- Desk
- Individually controlled air conditioning
- Wireless Internet access
Distances from Seamar HotelCeara Museum of Image and Sound - 1 km / 0.6 mi
CEART Artisan Center - 1 km / 0.6 mi
Ponte dos Ingleses - 1.2 km / 0.7 mi
Fortaleza Handicraft Market - 1.3 km / 0.8 mi
Dragao do Mar Art and Culture Center - 1.4 km / 0.9 mi
Maracatu Museum - 1.4 km / 0.9 mi
Bandeira Square - 1.4 km / 0.9 mi
Mini Siara Museum - 1.5 km / 1 mi
Portugal Square - 1.6 km / 1 mi
Aldeota Shopping Center - 1.7 km / 1 mi
INACE Shipyard - 1.8 km / 1.1 mi
Figueiras de Melo Square - 1.8 km / 1.1 mi
Central Market - 1.8 km / 1.1 mi
Se Cathedral - 1.9 km / 1.2 mi
Shopping Del Paseo - 1.9 km / 1.2 mi
The preferred airport for Seamar Hotel is Fortaleza (FOR-Pinto Martins Intl.) - 7.2 km / 4.5 mi.
Distances are calculated in a straight line from the property's location to the point of interest or airport and may not reflect actual travel distance.
Distances are displayed to the nearest 0.1 mile and kilometer.
Transportation services Seamar Hotel
- Free parking
We have the accommodation you need, all over the world
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- • Cavallino Hotels, Italy
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- • Wetzikon Hotels, Switzerland
- • Bathgate Hotels, United Kingdom
- • Hua Hin Hotels, Thailand
- • Ålbæk Hotels, Denmark


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Latest PTSD Stories
Results to be announced of Legion's online survey of veterans suffering from the conditions WASHINGTON, June 9, 2014 /PRNewswire-USNewswire/ -- Complementary and alternative treatments for
MIAMI, June 5, 2014 /PRNewswire-USNewswire/ -- Children exposed to large amounts of media coverage following a terrorist attack can develop serious mental health difficulties, regardless of
Mental Training is an integral part of any professional or Olympic athlete's daily routine.
OCONOMOWOC, Wis., May 20, 2014 /PRNewswire/ -- Physical or sexual assault, a car or other accident, a natural disaster or even witnessing a life-threatening event are only some of the events
Patients who have survived a stay in the intensive care unit (ICU) have a greatly increased risk of developing symptoms of post-traumatic stress disorder (PTSD)
K9s For Warriors trains shelter dogs and matches them with veterans suffering from post-traumatic stress disorder and traumatic brain injury SHAWNEE, Kan.
World-renowned mountaineers join injured soldiers to overcome barriers FORT COLLINS, Colo., April 30, 2014 /PRNewswire/ -- Soldiers to Summits, a program of No Barriers USA, and Mission: Mt.
With a new generation of military veterans returning home from Iraq and Afghanistan, post-traumatic stress disorder (PTSD) has become a prominent concern in American medical institutions and the culture at-large.
In the aftermath of war, communities who continue to experience repeated violence could have a major escalation in rates of post-traumatic stress disorder (PTSD) and severe distress.
Two Nonprofits Work Together to Strengthen Program Work for All Veterans Annandale, VA (PRWEB) April 18, 2014 Hope For The Warriors® (
Latest PTSD Reference Libraries
Posttraumatic stress disorder (commonly known as PTSD) is a severe anxiety disorder characterized by adverse anxiety-related experiences, behaviors, and physiological responses that results from exposure to an event that causes psychological trauma. Examples of events that could lead to PTSD include the threat of harm or death to oneself or to someone else, or witnessing an event that overwhelms the individual's ability to cope. Diagnostic symptoms for PTSD include flashbacks or nightmares,...
- Forsooth! indeed! originally a parenthetical phrase used in repeating the words of another with more or less contempt or disdain.


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John Lewis Azuki Bedding, Pink
Product information
Brighten up bedtime! Each vibrant stripe brings something new to this premium duvet cover, with a colour palette ranging from energetic fuchsia through to zesty citrus.
What makes this beautiful bed linen all the more indulgent is the detailed dobby weave; a lustrous technique that brings sumptuous texture and incredible smoothness to each band of bright colour.
- Standard pillowcase to fit 75cm x 50cm
- Square pillowcase to fit 65cm x 65cm
- Single duvet cover to fit 135cm x 200cm
- Double duvet cover to fit 200cm x 200cm
- Kingsize duvet cover to fit 230cm x 220cm
- Super kingsize duvet cover to fit 260cm x 220cm
- Brand
John Lewis
- Duvet Cover Style
- Stripe
- Eligible for International Delivery
- Iron
- Medium iron
- Ironing instructions
- Warm iron
- Material
- 100% cotton percale
- Pillowcases included?
- Pillowcases not included
- Thread count
- 200
- Tumble Dry
- Yes, at lower temperature
- Washing Instructions
- Machine washable at 40°C
Click & collectFree from John Lewis or Waitrose shops for collection tomorrow from 2pm
365 participating John Lewis and Waitrose shops
- Free
Collect + from a local shop from tomorrow - postcode exclusions apply for Collect+ deliveries5000+ participating Collect+ shops
- £3.00
Standard delivery in 5 working days
- £3.00
Named or Saturday UK delivery
- £6.95
Named day UK delivery pre-10.30am
- £9.95
International delivery available
- Europe (Non EU) - £7.50
- Europe (EU) - £7.50
- Rest of World - £25.00
- Australia - £25.00
- £7.50
We want you to be happy with your purchase. If you're not, depending on the type of product, you can make free returns in the UK via John Lewis shops, Royal Mail, Collect+, or MyHermes. If you need to arrange collection of large or heavy items, call us on 03456 049 049. For international returns queries, call us on +44 1698 545 454. Find out more about returns.
You'll find returns information on your delivery note. Unless faulty, we'd like you to make your return within 90 days of purchase. Exceptions apply to certain products - find out more about our refund policy.
Q & As
There's more to duvets, pillows and mattress protection than meets the eye
Choose the best bed linen and buy a good night's sleep too
Choose the best bed linen and buy a good night's sleep too
UK delivery:
£3.00 Standard delivery in 5 working days
Click & collect
Free Click & collect from John Lewis or Waitrose shops for collection tomorrow from 2pm
£3.00 Collect+ from a local shop from tomorrow
International delivery available:
From £7.50


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
31 year old man | Burr Ridge, IL, USA | Seeking women 26-33 within 30 miles
Active within 5 days
Heard great things about online dating.
I come from a big Italian family that I am very close with and wouldn't want it any other way. I am a very loyal, funny. I think I am shy at first but once I get to know someone I enjoy opening up. Very close to my brother, sister and my parents and would like a women with great family values as well. I enjoy trying new restaurants and love italian food. Also like the relaxing nights at home with a movie and dinner. Like to hang out with my friends. A few years back I switched my career from Architecture to retail management and I really love my job. I try to use my architecture background every chance I get at my job. I am very happy that a fell into a great company with lots of room for advancement. As for what I am looking for is someone who is loyal, loving and caring with great family values. Message me any other questions and I will be happy to answer. Hope to talk to you soon
Business networking , Cooking , Dining out , Movies/Videos , Museums and art , Music and concerts , Playing cards , Playing sports , Travel/Sightseeing , Watching sports , Wine tasting
Baseball, Billiards / Pool, Bowling, Football, Golf, Hockey, Swimming, Weights / Machines
I exercise 1-2 times per week.
I like Dogs
Middle of the Road
Downtown Chicago, Vegas, Florida, Cancun. Northern Michigan, Nashville.
DePaul University, Chicago, IL
Music: I like basically all music except for heavy metal. Tv shows: How I met your mother, Two and a Half Men, Banshee, Revolution, Chicago Fire, Chicago PD,
Going to or watching Blackhawks, Cubs and Bears game. Or watching any hockey game. Hanging out with friends and family. I enjoy going to the clubs in chicago just not all the time. Going to movies. Sitting outside
News and sports online everyday. Beer books and Whiskey books for work
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'5" (165cms)
|3'0" (91cms) to 5'4" (162cms)
|Body type:
|About average
|Slender, About average, Athletic and toned
|No preference
|Dark brown
|Black, Light brown, Dark brown, Blonde, Dark blonde, Platinum
|No Way
|No Way, Occasionally, Daily, Yes, but trying to quit
|Social Drinker
|Social Drinker, Moderately
|Retail / Food services
|No preference
I used to work in a small architecture firm while I was in the process of getting my masters degree. That was about four years ago since the economy got bad the company I worked for went out of business. Now in retail management.
|I'll tell you later
|No preference
|Never Married
|Never Married
|Have kids:
|Wants kids:
|White / Caucasian
|White / Caucasian
75% Italian and 25% Irish
|Christian / Catholic
|Christian / Catholic
|Bachelors degree
|High school