Protein Expression and Purification

Appraisal of translocation pathways for displaying ankyrin repeat protein on phage particles.

Publication Date: 2010 Aug 24 PMID: 20800093
Authors: Nangola, S. - Minard, P. - Tayapiwatana, C.
Journal: Protein Expr Purif

Depending on the molecular properties of the proteins of interest (POI), the rate of success in displaying proteins on phage particles is unpredictable. Formation of polypeptide tertiary structure in the cytoplasm occasionally results in low level display on viral particles. Here we assessed the influence of different leader peptides on the display of a premature cytoplasmic folding protein, ankyrin repeat protein (ARP), via the minor coat protein pIII. These peptides include the Sec, SRP and Tat pathways. The results demonstrated that the Sec and SRP pathways were capable of displaying the protein on the viral particle, whereas the Tat pathway failed to do so. Interestingly, the Tat pathway efficiently directed ARP through its translocon without fusing with pIII. Furthermore, the soluble form of ARP was detected in Escherichia coli periplasm.

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Effect of IPTG amount on apo- and holo- forms of glycerophosphate oxidase expressed in Escherichia coli.

Publication Date: 2010 Aug 22 PMID: 20736068
Authors: Zheng, C. - Zhao, Z. - Li, Y. - Wang, L. - Su, Z.
Journal: Protein Expr Purif

Escherichia coli has proved to be a successful host for the expression of many heterologous proteins, and much efforts have been made toward improving recombinant protein expression including the usage of strong promoters and co-expression with chaperones. But little attention was paid on the relation between expression level and function of the target protein. Glycerophosphate oxidase (GPO) is a protein with FAD cofactor (without free cysteine and disulfide bonds).It was observed that the specific activity of GPO dramatically decreased with the increase of inducer IPTG. In addition, the stability of it decreased correspondingly. The structural difference of samples expressed under varying IPTG was investigated using size-exclusion and reverse-phase high performance liquid chromatography, together with CD spectrum. It was found that the conformation of peptide and organization of subunits were not affected. The loss of specific activity and stability were correlated to incomplete attachment of FAD onto GPO. These results revealed that synthesis speed should be controlled either by reduction of IPTG amount or using weak promoters in the production of GPO.

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Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker.

Publication Date: 2010 Aug 21 PMID: 20732425
Authors: Bradley, L. H. - Bricken, M. L. - Randle, C.
Journal: Protein Expr Purif

Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.

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A SUMO-Groucho Q domain fusion protein: Characterization and in vivo Ulp1-mediated cleavage.

Publication Date: 2010 Aug 21 PMID: 20732424
Authors: Kuo, D. - Nie, M. - De Hoff, P. - Chambers, M. - Phillips, M. - Hirsch, A. M. - Courey, A. J.
Journal: Protein Expr Purif

We describe here a system for the expression and purification of small ubiquitin-related modifier (SUMO) fusion proteins, which often exhibit dramatically increased solubility and stability during expression in bacteria relative to unfused proteins. The vector described here allows expression of a His-tagged protein of interest fused at its N-terminus to SUMO. Using this vector, we have produced a polypeptide consisting of SUMO fused to the Q domain of Drosophila Groucho in a concentrated soluble form. Hydrodynamic analysis shows that, consistent with previous studies on full-length Groucho, the fusion protein forms an elongated tetramer, as well as higher order oligomers. After expressing a protein as a fusion to SUMO, it is often desirable to cleave the SUMO off of the fusion protein using a SUMO-specific protease such as Ulp1. To facilitate such processing, we have constructed a dual expression vector encoding two fusion proteins: one consisting of SUMO fused to Ulp1 and a second consisting of SUMO fused to a His-tagged protein of interest. The SUMO-Ulp1 cleaves both itself and the other SUMO fusion protein in the bacterial cells prior to lysis, and the proteins retain solubility after cleavage.

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Molecular manipulation associated with disulfide bond formation to enhance the stability of recombinant therapeutic protein.

Publication Date: 2010 Aug 16 PMID: 20719248
Authors: Zhang, L. - Moo-Young, M. - Chou, C. P.
Journal: Protein Expr Purif

Cys(27) in the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein, was identified as a target for molecular manipulation. Two Escherichia coli strains of BL21(DE3) and Origami B(DE3), respectively, with a reducing and an oxidative cytoplasm were used as the expression host to produce the Cys(27) mutants. It was observed that Cys(27) was involved in the in vivo formation of intramolecular disulfide bonds when hCD83ext was expressed in Origami B(DE3). The Origami-derived protein products had a higher tendency than the BL21-derived counterparts for multimerization via the in vitro formation of intermolecular disulfide bonds. Various analyses were conducted to identify the structural differences among these mutant variants. Most importantly, molecular stability was enhanced by the Cys(27) mutations since the Cys(27) mutants derived from either BL21 or Origami were much less susceptible to degradation compared to wild-type hCD83ext. This study highlights the implications of aberrant disulfide bond formation on the production of therapeutic proteins.

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Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: Using structural genomics tools for a challenging target.

Publication Date: 2010 Aug 15 PMID: 20716446
Authors: Lee, T. V. - Lott, J. S. - Johnson, R. D. - Arcus, V. L.
Journal: Protein Expr Purif

Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination.

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Production and purification of human papillomavirus type 33 L1 virus-like particles from Spodoptera frugiperda 9 cells using two-step column chromatography.

Publication Date: 2010 Aug 15 PMID: 20716445
Authors: Baek, J. O. - Seo, J. W. - Kim, I. H. - Kim, C. H.
Journal: Protein Expr Purif

The major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. The gene encoding the major capsid protein L1 of the high-risk HPV type 33 was isolated from a Korean patient and expressed in Sf-9 insect cells using a baculovirus expression system. HPV33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs, and we thus used this system. The expression of HPV L1 protein in Sf-9 cells was examined by SDS-PAGE, Western-blotting, and ELISA analyses, and the data showed that HPV33 L1 VLPs were determined to >98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV VLPs were approximately 50-60nm in diameter and created by self-assembly of HPV L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine.

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High-yield production and characterization of biologically active GST-tagged human topoisomerase IIalpha protein in insect cells for the development of a high-throughput assay.

Publication Date: 2010 Aug 13 PMID: 20709174
Authors: Singh, P. K. - Chan, P. - Hibbs, M. J. - Vazquez, M. J. - Segura, D. C. - Thomas, D. A. - Theobald, A. J. - Gallagher, K. T. - Hassan, N. J.
Journal: Protein Expr Purif

DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase IIalpha proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase IIalpha using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40mg/L i.e. 8mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase IIalpha was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase IIalpha inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC50 values obtained were in good agreement with published data. These inhibitors also demonstrated 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.

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Activation by phosphorylation and purification of human c-Jun N-terminal kinase (JNK) isoforms in milligram amounts.

Publication Date: 2010 Aug 13 PMID: 20709173
Authors: Thevenin, A. F. - Zony, C. L. - Bahnson, B. J. - Colman, R. F.
Journal: Protein Expr Purif

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) signaling cascade. They are activated through dual phosphorylation of two residues in the activation loop, a threonine and a tyrosine, by MAP2 kinases (MKK4 and 7) in response to various extracellular stresses such as UV or osmotic shock, as well as by cytokines and growth factors. Only small amounts of phosphorylated, active JNKs have previously been produced because of difficulties in expressing these phosphorylated kinases in Escherichia coli, which lack the appropriate upstream kinases. We have now established a novel activation and purification method that allows for reproducible production of milligram amounts of active, phosphorylated JNKs suitable for a variety of enzymatic, biophysical and structural characterizations. We utilize N-terminally His-tagged MKK4 that is coexpressed in E. coli with a constitutively active form of MEKK1. This phosphorylated, active His-MKK4 is purified by Ni-NTA chromatography and used to phosphorylate milligram amounts of three different isoforms of human JNKs (JNK1alpha1, JNK1alpha2 and JNK2alpha2) that had separately been expressed and purified from E. coli in their inactive forms. These in vitro activated JNKs are phosphorylated on both residues (T183, Y185) in their activation loops and are active towards their substrate, ATF2.

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Expression of cellobiose dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization.

Publication Date: 2010 Aug 13 PMID: 20709172
Authors: Zhang, R. - Fan, Z. - Kasuga, T.
Journal: Protein Expr Purif

A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a His(6)-tag (rNC-CDH1) was successfully expressed and secreted. rNC-CDH1 was produced at the level of 652IU/L after 2days of cultivation in the induction medium. The His(6)-tagged rNC-CDH1 was purified through a one-step Ni-NTA affinity column under non-denaturing conditions. The purified rNC-CDH1 has a CDH activity of 7451IU/L (0.89mg protein/mL), with a specific CDH activity of 8.37IU/mg. The purity of the enzyme was examined by SDS-PAGE, and a single band corresponding to a molecular weight of about 120kDa was observed. Activity staining confirmed the CDH activity of the protein band. The purified rNC-CDH1 has maximum CDH activity at pH 4.5, and a rather broad temperature optimum of 25-70 degrees C. Kinetic analysis showed cellobiose and cellooligosaccharides are the best substrates for rNC-CDH1. The K(m) value of the rNC-CDH1 for cellooligosaccharide increases with the elongation of glucosyl units. k(cat) remains relatively constant when the chain length changes.

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Production of protein complexes via co-expression.

Publication Date: 2010 Aug 6 PMID: 20692346
Authors: Kerrigan, J. J. - Xie, Q. - Ames, R. S. - Lu, Q.
Journal: Protein Expr Purif

Multi-protein complexes are involved in essentially all cellular processes. A protein's function is defined by a combination of its own properties, its interacting partners, and the stoichiometry of each. Depending on binding partners, a transcription factor can function as an activator in one instance and a repressor in another. The study of protein function or malfunction is best performed in the relevant context. While many protein complexes can be reconstituted from individual component proteins after being produced individually, many others require co-expression of their native partners in the host cells for proper folding, stability, and activity. Protein co-expression has led to the production of a variety of biological active complexes in sufficient quantities for biochemical, biophysical, structural studies, and high throughput screens. This article summarizes examples of such cases and discusses critical considerations in selecting co-expression partners, and strategies to achieve successful production of protein complexes.

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Bordetella pertussis CyaA-RTX subdomain requires calcium ions for structural stability against proteolytic degradation.

Publication Date: 2010 Aug 4 PMID: 20691271
Authors: Pojanapotha, P. - Thamwiriyasati, N. - Powthongchin, B. - Katzenmeier, G. - Angsuthanasombat, C.
Journal: Protein Expr Purif

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming (CyaA-PF) domain expressed in Escherichia coli was shown to retain its hemolytic activity. Here, a 100-kDa RTX (Repeat-in-ToXin) subcloned fragment (CyaA-RTX) containing a number of putative calcium-binding repeats was further investigated. The recombinant CyaA-RTX protein, although expressed as a soluble form in a protease-deficient E. coli strain BL21(DE3)pLysS, was found to be highly sensitive to proteolytic degradation. Interestingly, the addition of calcium ions in a millimolar range into the CyaA-RTX preparation significantly prevented the degradation. Moreover, levels of proteolytic degradation were dependent on calcium concentrations, implying an important role for calcium-binding sites in the RTX subdomain for structural stability. Homology-based modeling of the repetitive blocks in the CyaA-RTX subdomain supports that this calcium-bound protein folds into a parallel beta-roll structure with calcium ions acting as a structural stabilizing bridge.

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Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants.

Publication Date: 2010 Aug 4 PMID: 20691270
Authors: Kim, T. G. - Kim, M. Y. - Yang, M. S.
Journal: Protein Expr Purif

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB-EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB-EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB-EIII fusion protein in transgenic plant extracts. Based on the G(M1)-ELISA results, the CTB-EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, G(M1)-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB-EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.

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Expression, purification, and refolding of a recombinant human bone morphogenetic protein 2 in vitro.

Publication Date: 2010 Aug 4 PMID: 20691269
Authors: Zhang, Y. - Ma, Y. - Yang, M. - Min, S. - Yao, J. - Zhu, L.
Journal: Protein Expr Purif

In this work, the recombinant human bone morphogenetic protein 2 (rhBMP-2) gene was cloned from MG-63 cells by RT-PCR, and the protein was expressed in Escherichia coli expression system, purified by Ni-NTA column under denaturing conditions and refolded at 4 degrees C by urea gradient dialysis. We found that the protein refolding yield was increased with the increase of pH value from pH 6.0 to pH 9.0. The yield was 42% and 96% at pH 7.4 and pH 9.0, respectively, while that at pH 6.0 was only 3.4%. The cell culture results showed that the rhBMP-2 refolded at pH 7.4 urea gradient dialysis had higher biological activity for MG-63 cell proliferation and differentiation than that refolded at pH 9.0 since pH 7.4 is closer to the conditions in vivo leading to the formation of dimers through the interchain disulfide bond. Moreover, the biological activity for MG-63 was promoted with the increase of rhBMP-2 concentration in the cell culture medium. This work may be important for the in vitro production and biomedical application of rhBMP-2 protein.

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Production of soluble, active acetyl serotonin methyl transferase in Leishmania tarentolae.

Publication Date: 2010 Aug 3 PMID: 20688166
Authors: Ben-Abdallah, M. - Bondet, V. - Fauchereau, F. - Beguin, P. - Goubran-Botros, H. - Pagan, C. - Bourgeron, T. - Bellalou, J.
Journal: Protein Expr Purif

N-acetyl serotonin methyl transferase (ASMT) is the last enzyme in the melatonin synthesis pathway. Evidence linking autism-related disorders with disorders of melatonin metabolism, and, more specifically, with mutations of the gene encoding ASMT, prompted us to investigate the properties and localization of this enzyme. As a first step, we undertook to overproduce the protein in a recombinant host. Early attempts to produce ASMT in recombinant Escherichia coli yielded only insoluble and heavily degraded material. However, recombinant ASMT (rASMT) could be produced in soluble, active form and purified in milligram amounts when the gene was cloned and expressed in Leishmania tarentolae.

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High-level expression and reconstitution of active Cfr, a radical-SAM rRNA methyltransferase that confers resistance to ribosome-acting antibiotics.

Publication Date: 2010 Aug 3 PMID: 20678576
Authors: Booth, M. P. - Challand, M. R. - Emery, D. C. - Roach, P. L. - Spencer, J.
Journal: Protein Expr Purif

Cfr is a radical-SAM (S-adenosyl-l-methionine) enzyme that methylates the 8 position of 23S rRNA residue A2503 to confer resistance to multiple antibiotic classes acting upon the large subunit of the bacterial ribosome. Radical-SAM enzymes use an Fe-S cluster to generate the 5'-deoxyadenosyl (DOA) radical from SAM, enabling them to modify intrinsically unreactive centres such as adenosine C8. However, despite its mechanistic interest and clinical relevance, until recently Cfr remained little characterised. Accordingly we have used co-expression with the Azotobacter vinelandii isc operon, encoding genes responsible for Fe-S cluster biosynthesis, to express hexahistidine-tagged Cfr in Escherichia coli BL21Star, and purified the recombinant protein in a yield more than 20times greater than has been previously reported. As aerobically purified, Cfr contains secondary structure, is monomeric in solution and has an absorbance spectrum suggestive of a 2Fe-2S cluster. After anaerobic purification a 4Fe-4S cluster is indicated, while on reconstitution with excess iron and sulphide a further increase in metal content suggests that an additional, most likely 4Fe-4S, cluster is formed. Acquisition of additional secondary structure under these conditions indicates that Fe-S clusters are of structural, as well as functional, importance to Cfr. In the presence of sodium dithionite reconstituted Cfr is both reducible and able to cleave SAM to 5'-deoxyadeonsine (DOA), demonstrating that the purified reconstituted enzyme has radical-SAM activity. Co-expression with isc proteins thus enables recombinant active Cfr to be obtained in yields that facilitate its future spectroscopic and structural characterisation.

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Purification of functional human Cl(-)/HCO(3)(-) exchanger, AE1, over-expressed in Saccharomyces cerevisiae.

Publication Date: 2010 Nov PMID: 20609390
Authors: Bonar, P. - Casey, J. R.
Journal: Protein Expr Purif

There is no high-resolution structure for the membrane domain of the human erythrocyte anion exchanger, AE1 (Band 3). In this report, we have developed an expression and purification strategy for AE1 to be used in crystallization trials. Saccharomyces cerevisiae strain BJ5457 was transformed with an expression vector encoding the AE1 membrane domain (AE1MD, amino acids 388-911), fused C-terminally to an epitope tag, corresponding to the nine C-terminal amino acids of rhodopsin. The fusion protein, AE1MD-Rho, was expressed at a concentration of 0.3 mg/l of culture. Confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation revealed that AE1MD-Rho did not process to the plasma membrane of S. cerevisiae, but was retained in an intracellular membrane fraction. Treatment with the endoglycosidase, PNGase F, showed that AE1MD-Rho is not N-glycosylated. AE1MD-Rho solubilized from yeast membranes, with Fos-choline detergent, was purified to 93% homogeneity in a single-step, using a 1D4 antibody affinity resin, in amounts up to 2.5 mg from 18 l of culture. The ability of purified AE1MD-Rho to transport sulfate was examined in reconstituted vesicles. The rate of sulfate efflux mediated by vesicles reconstituted with AE1MD-Rho was indistinguishable from vesicles with purified erythrocyte-source AE1. Using this purification strategy, sufficient amounts of functional, homogeneous AE1MD-Rho can be purified to enable crystallization trials.

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Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography.

Publication Date: 2010 Nov PMID: 20600950
Authors: Tan, L. C. - Chua, A. J. - Goh, L. S. - Pua, S. M. - Cheong, Y. K. - Ng, M. L.
Journal: Protein Expr Purif

Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent. Here, we describe the use of metal affinity membrane chromatography as a rapid and improved alternative for the purification of recombinant DIII (rDIII) antigens from DENV serotypes 1-4 and WNV - New York, Sarafend, Wengler and Kunjin strains. Optimum conditions for the expression, solubilization, renaturation and purification of these proteins were established. The purified proteins were confirmed by MALDI-TOF mass spectrometry and ELISA using antibodies raised against the respective viruses. Biological function of the purified rDIII proteins was confirmed by their ability to generate DIII-specific antibodies in mice that could neutralize the virus.

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A series of bacterial co-expression vectors with rare-cutter recognition sequences.

Publication Date: 2010 Nov PMID: 20600949
Authors: Wakamori, M. - Umehara, T. - Yokoyama, S.
Journal: Protein Expr Purif

The bacterial co-expression method is a useful protein expression technique to reconstitute a hetero-oligomeric protein complex in vitro. However, during the plasmid subcloning for co-expression, unintended cleavage at the sequences of target cDNAs becomes more frequent as the number of DNA inserts increases. This problem also makes it difficult to change the combination of targeted proteins after preparing a certain co-expression construct. To avoid this problem, we have developed a series of bacterial co-expression vectors, in which each translation cassette can be subcloned at a set of rare-cutter restriction enzyme sites. We selected 9 different rare-cutter restriction enzymes that recognize a 7 or 8-base-pair sequence, and constructed 27 kinds of cloning vectors and 3 kinds of co-expression vectors, utilizing the rare-cutter recognition sequences as multi-cloning sites. Using this vector system, we co-expressed and co-purified a 7-subunit protein complex composed of the mammalian 26S proteasome regulatory subunits RPT1 to RPT6, and their associated factor, gankyrin. We verified the presence of all 7 subunits by western blotting, by taking advantage of the vector system in which the target proteins can be fused with a broad repertoire of epitope tags.

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Heterologous high yield expression and purification of neurotensin and its functional fragment in Escherichia coli.

Publication Date: 2010 Nov PMID: 20600945
Authors: Tapaneeyakorn, S. - Ross, S. - Attrill, H. - Watts, A.
Journal: Protein Expr Purif

Peptide synthesis is widely used for the production of small proteins and peptides, but producing uniformly isotopically labelled peptides for NMR and other biophysical studies could be limited for economic reasons. Here, we propose a use of a modified pGEV-1 plasmid to express neurotensin (NT(1-13)), pGlu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(10)-Ty r(11)-Ile(12)-Leu(13)-OH, as a C-terminal fusion protein with the GB1 domain of streptococcal protein G. The free carboxyl-terminus is important for the function of several peptide hormones, including neurotensin. Therefore, for the pGEV-NT(1-13) construct, the C-terminal pGEV-encoded 6xHis tag was removed and an N-terminal 8xHis tag was introduced for affinity purification. To facilitate removal of tags using CNBr cleavage, a methionine was introduced at the N-terminal of the peptide. Furthermore, this pGEV-NT(1-13) plasmid was used as a template to include a Pro-7 to Met mutation for CNBr cleavage, giving NT(8-13), the sub-fragment crucial for the biological activity of this peptide. These two constructs are being used to produce uniformly labelled NT(1-13) and NT(8-13) in high yield and in a cost effective way, using cheap (15)N and/or (13)C source. The modification proposed here using the pGEV-1 plasmid could be an alternative option for the high expression of other isotopically labelled and unlabelled short peptides, including hormones and hydrophobic membrane peptides.

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Enhancing the secretory yields of leech carboxypeptidase inhibitor in Escherichia coli: influence of trigger factor and signal recognition particle.

Publication Date: 2010 Nov PMID: 20600941
Authors: Puertas, J. M. - Nannenga, B. L. - Dornfeld, K. T. - Betton, J. M. - Baneyx, F.
Journal: Protein Expr Purif

The signal recognition particle (SRP) dependent secretion pathway is as an attractive alternative to Sec-dependent export for the production of disulfide-bonded and/or fast-folding recombinant proteins in the Escherichia coli periplasm. SRP, which shares a ribosomal attachment site with the molecular chaperone trigger factor (TF), recognizes highly hydrophobic signal sequence as they emerge from the ribosome and delivers ribosome nascent chain complexes to FtsY for subsequent cotranslational translocation of target proteins across the SecYEG pore. However, like in the case of Sec-dependent export, secretory yields can be limited by the accumulation of precursor proteins in the cytoplasm. Using leech carboxypeptidase inhibitor (LCI) fused to the SRP-dependent DsbA signal sequence as a model system, we show that a null mutation in the gene encoding TF (Deltatig) or SRP co-expression reduce pre-LCI accumulation by half, and that quantitative export can be achieved by combining the two strategies. Interestingly, enhanced precursor processing did not alter periplasmic LCI levels but increased the amount of protein excreted in the growth medium. All mature LCI was nearly fully active and an 80% increase in productivity was achieved in Deltatig cells alone due to their faster growth. Our results show that competition between SRP and TF can interfere with efficient export of recombinant proteins targeted to the SRP pathway and establish TF-deficient strains and SRP co-expression as a simple solution to improve yields.

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Human papillomavirus-like particles vaccine efficiently produced in a non-fermentative system based on insect larva.

Publication Date: 2010 Nov PMID: 20600940
Authors: Millan, A. F. - Gomez-Sebastian, S. - Nunez, M. C. - Veramendi, J. - Escribano, J. M.
Journal: Protein Expr Purif

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Purification of high yields of catalytically active lysyl oxidase directly from Escherichia coli cell culture.

Publication Date: 2010 Nov PMID: 20600936
Authors: Herwald, S. E. - Greenaway, F. T. - Lopez, K. M.
Journal: Protein Expr Purif

Lysyl oxidase is a highly insoluble enzyme requiring high concentrations of urea to solubilize. A method to obtain lysyl oxidase in high yields directly from an Escherichia coli culture without the need for refolding of inclusion bodies has been developed using nutrient rich media. pET21b was used to overexpress the lysyl oxidase enzyme and to introduce a C-terminal 6X histidine tag for purification. Lysyl oxidase yields of 10 mg of active and properly folded enzyme per liter of media have been obtained. Purification was achieved via affinity chromatography using a Ni-NTA column. Copper content was found to be 19%. LTQ cofactor formation in LOX is a self-processing event in the presence of copper. LTQ content was determined to be 24% based on reaction with phenylhydrazine to form a phenylhydrazone adduct. Quantification of this adduct was attained using the previously reported extinction coefficient of 15.4 mM(-1)cm(-1). LTQ presence was also verified by redox cycling. Specific enzymatic activity was measured to be 0.31 U/mg, one of the highest activities reported.

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Production and purification of the penicillin-binding protein 3 from Pseudomonas aeruginosa.

Publication Date: 2010 Oct PMID: 20580675
Authors: de Leon, S. R. - Daniels, K. - Clarke, A. J.
Journal: Protein Expr Purif

The penicillin-binding proteins (PBPs) are peripheral membrane enzymes that catalyze the final steps for the biosynthesis of the essential bacterial cell wall heteropolymer peptidoglycan. Bacteria produce a number and variety of PBPs which are classified as either high molecular weight or low molecular weight PBPs. The high molecular weight PBPs are multimodular being comprised of an N-terminal membrane anchor followed by a non-penicillin binding domain and a C-terminal penicillin-binding domain. The penicillin-binding domain functions as a serine-acyl transpeptidase to catalyze the crosslinking of neighboring glycan strands within the peptidoglycan sacculus. PBP 3 from Escherichia coli has been studied extensively and it has been shown to be responsible for the synthesis of peptidoglycan during the division and septation of the cells. The opportunistic human pathogen Pseudomonas aeruginosa produces a similar compliment of PBPs to E. coli, but differences in their organization and function have been noted. To investigate these differences further, appropriate quantities of each of the P. aeruginosa PBPs are required in forms amenable for study both in vivo and in vitro. Herein, we describe the cloning and expression of the ftsI gene encoding PBP 3from P. aeruginosa. The PBP was engineered in soluble form to facilitate its study in vitro and with a hexa-His tag to permit its facile purification by affinity chromatography. The recombinant proteins were demonstrated to bind penicillin and these forms of the PBPs were shown to be useful in studying their localization within their host cells by immunogold transmission electron microscopy.

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Expression of four mutant fibrinogen gammaC domains in Pichia pastoris confirms them as causes of hypofibrinogenaemia.

Publication Date: 2010 Oct PMID: 20580674
Authors: Sheen, C. R. - Dear, A. - Brennan, S. O.
Journal: Protein Expr Purif

Mutations in the fibrinogen gene cluster can cause low plasma fibrinogen concentrations, known as hypofibrinogenaemia. It is important to verify whether a detected sequence variant in this cluster is deleterious or benign and this can be accomplished using protein expression systems. In this study, four mutations in the fibrinogen gammaC domain that had previously been described in patients with hypofibrinogenaemia were introduced into a gammaC construct and expressed in a Pichia pastoris yeast system to investigate their effects on protein stability and secretion. These experiments showed that the fibrinogen Middlemore (N230D), Dorfen (A289V), Mannheim II (H307Y), and Muncie (T371I) mutations were not secreted, supporting their causative role in hypofibrinogenaemia. Overexpression of the N230D, A289V and H307Y mutants revealed that the majority of the synthesised protein was retained in the endoplasmic reticulum, with only a minor proportion reaching the trans-Golgi network. Regardless, none of this protein was secreted which confirms that the four mutations investigated are indeed responsible for hypofibrinogenaemia.

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Efficient production of human Fas receptor extracellular domain-human IgG1 heavy chain Fc domain fusion protein using baculovirus/silkworm expression system.

Publication Date: 2010 Oct PMID: 20576530
Authors: Muraki, M. - Honda, S.
Journal: Protein Expr Purif

The fusion protein consisting of human Fas receptor extracellular domain and human IgG1 heavy chain Fc domain (hFasRECD-Fc) is a medically important protein that potentially has therapeutic uses. The fusion gene composed of a synthetic human Fas receptor extracellular domain gene and the cDNA encoding human IgG1 heavy chain Fc domain was investigated on the secretory expression using two baculovirus systems which employed either Spodoptera frugiperda 9 (Sf9) cell line or Bombyx mori (silkworm) larvae as the host organism. Both expression systems produced the functional hFasRECD-Fc as a dimer molecule linked by disulfide bridges. The secretion level per unit volume was much higher in the case of silkworm larvae as compared to Sf9 cell line, and was estimated to be more than 150 times. A substantially pure hFasRECD-Fc sample from silkworm larvae was obtained by single step Protein G-agarose affinity column chromatography. The affinity purified sample was further fractionated by anion-exchange chromatography with the final purification yield of 22.5 mg from 26 ml hemolymph. The hFasRECD-Fc from silkworm larvae and the tag-free human Fas ligand extracellular domain derivative from Pichia pastoris formed a stable complex in solution, which was verified by size-exclusion chromatography. This study demonstrated that the baculovirus/silkworm expression system provided the means for efficient production of highly pure hFasRECD-Fc with functional activity.

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Purification and characterization of Stn1p, a single-stranded telomeric DNA binding protein.

Publication Date: 2010 Oct PMID: 20576529
Authors: Qian, W. - Fu, X. H. - Zhou, J. Q.
Journal: Protein Expr Purif

In Saccharomyces cerevisiae, Stn1p and Ten1p are required for telomere maintenance. These two proteins and another telomeric single-stranded DNA binding protein, Cdc13p, have been proposed to form a complex to control telomere integrity. In this work, we purified the recombinant Stn1p in Escherichia coli and found that the purified protein could specifically interact with single-stranded telomeric DNA in vitro. Co-fractionation of co-overexpressed Stn1p and Ten1p in insect cells revealed their stable association. A Stn1p/Ten1p binary complex was reconstituted with purified recombinant proteins in vitro. These results indicated that Stn1p and Ten1p interact with each other directly, which is important in telomere length regulation and end protection.

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Expression and purification of two different antimicrobial peptides, PR-39 and Protegrin-1 in Escherichia coli.

Publication Date: 2010 Oct PMID: 20573563
Authors: Fan, F. - Wu, Y. - Liu, J.
Journal: Protein Expr Purif

To implement coexpression of antimicrobial peptides PR-39 and Protegrin-1 (PG-1) in prokaryotic expression system, a tandem gene fragment encoding PR-39 and PG-1 has been synthesized chemically. The cleavage site (Asn-Gly) of hydroxylamine hydrochloride was introduced between PR-39 and PG-1. The fragment was inserted into vector pGEX-4T-1 and expressed in Escherichia coli. The fusions of single peptides to GST were created at the same time. The fusion protein GST-PR-39-PG-1, purified by affinity chromatography, was cleaved first by hydroxylamine hydrochloride to release recombinant PG-1 and then by enterokinase to release PR-39. Purification of recombinant PR-39 and PG-1 was achieved. About 1.9 mg/l recombinant PR-39 and 1.1 mg/l PG-1 were obtained. The recombinant antimicrobial peptides showed antibacterial activities that were similar to those released from fusions of single peptides to GST.

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Expression of neurotransmitter transporters for structural and biochemical studies.

Publication Date: 2010 Oct PMID: 20566324
Authors: Elbaz, Y. - Danieli, T. - Kanner, B. I. - Schuldiner, S.
Journal: Protein Expr Purif

Neurotransmitter transporters play essential roles in the process of neurotransmission. Vesicular neurotransmitter transporters mediate storage inside secretory vesicles in a process that involves the exchange of lumenal H(+) for cytoplasmic transmitter. Retrieval of the neurotransmitter from the synaptic cleft catalyzed by sodium-coupled transporters is critical for the termination of the synaptic actions of the released neurotransmitter. Our current understanding of the mechanism of these transporters is based on functional and biochemical characterization but is lacking high-resolution structural information. Very few structures of membrane transport systems from mammalian origin have been solved to atomic resolution, mainly because of the difficulty in obtaining large amounts of purified protein. Development of high yield heterologous expression systems suitable for mammalian neurotransmitter transporters is essential to enable the production of purified protein for structural studies. Such a system makes possible also the production of mutants that can be used in biochemical and biophysical studies. We describe here a screen for the expression of the vesicular monoamine transporter 2 (VMAT2) in cell-free and baculovirus expression systems and discuss the expression of VMAT2 in other systems as well (bacterial, yeast and mammalian cell lines). After screening and optimization, we achieved high yield (2-2.5 mg/l) expression of functional VMAT2 in insect cells. The system was also used for the expression of three additional plasma membrane neurotransmitter transporters. All were functional and expressed to high levels. Our results demonstrate the advantages of the baculovirus expression system for the expression of mammalian neurotransmitter transporters in a functional state.

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Structural identification of recombinant human CD83 mutant variant as a potent therapeutic protein.

Publication Date: 2010 Oct PMID: 20566323
Authors: Zhang, L. - Narayanan, N. - Brand, S. R. - Nicolette, C. A. - Baroja, M. - Arp, J. - Wang, H. - Moo-Young, M. - Chou, C. P.
Journal: Protein Expr Purif

The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein. In certain instances, hCD83ext protein products, even when stored frozen, tended to dimerize or even multimerize through the formation of aberrant intermolecular disulfide bonds. Herein, we discovered an analytical inconsistency and applied a modified sample preparation protocol for proper structural analysis of hCD83ext products which are heterologously expressed in Escherichia coli and subsequently purified. In addition, a mutant derivative with the Cys100Ser mutation was identified as an improved version which did not form dimers or multimers. The identification of this mutant variant as a more potent therapeutic protein than other hCD83ext species demonstrated that the structural variation associated with disulfide bond formation can be a critical issue for rigorous control of the quality and bioactivity of therapeutic proteins. The application of this mutant variant for protein therapeutics is currently under exploration.

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Expression, purification, and characterization of soluble K-Ras4B for structural analysis.

Publication Date: 2010 Oct PMID: 20566322
Authors: Abraham, S. J. - Muhamed, I. - Nolet, R. - Yeung, F. - Gaponenko, V.
Journal: Protein Expr Purif

A p21 GTPase K-Ras4B plays an important role in human cancer and represents an excellent target for cancer therapeutics. Currently, there are no drugs directly targeting K-Ras4B. In part, this is due to the lack of structural information describing unique features of K-Ras4B. Here we describe a methodology allowing production of soluble, well-folded K-Ras4B for structural analysis. The key points in K-Ras4B preparation are low temperature expression and extraction of K-Ras4B from the insoluble fraction using a nucleotide loading procedure in the presence of Mg(2+) and citrate, a low affinity chelator. Additionally, a significant amount of K-Ras4B could be extracted from the soluble fraction. We show that recombinant K-Ras4B is monomeric in solution. Excellent NMR signal dispersion suggests that the protein is well-folded and is amenable to solution structure determination. In addition, using phospholipid bilayer nanodiscs we show that recombinant K-Ras4B interacts with lipids and that this interaction is mediated by the C-terminal hypervariable region.

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Over-production of various secretory-form proteins in Streptomyces lividans.

Publication Date: 2010 Oct PMID: 20546899
Authors: Noda, S. - Ito, Y. - Shimizu, N. - Tanaka, T. - Ogino, C. - Kondo, A.
Journal: Protein Expr Purif

Streptomyces lividans is known to produce large amounts of proteins in culture supernatants. In this report, to expand the secretory expression system with a strong promoter derived from phospholipase D of Streptoverticillium cinnamoneum, we expressed three kinds of proteins: transglutaminase from Stv. cinnamoneum (StvcMTG) and beta-1,4-endoglucanase and beta-glucosidase from Thermobifida fusca YX. The StvcMTG gene was introduced into S. lividans using the shuttle vector pUC702 for Escherichia coli and S. lividans, and high level secretory production of StvcMTG (230 microg/ml in the culture supernatant) was achieved. The other prokaryotic proteins, beta-1,4-endoglucanase and beta-glucosidase, were also expressed in (His)(6)-tag fused form into culture supernatants and retained high activity. Furthermore, complete purification was achieved by conventional column or affinity column chromatography for each recombinant protein with 1 mg/ml over protein concentration. Three independent proteins were thus successfully expressed and purified, and we expect to use this system for the expression of other valuable heterologous proteins.

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The production, characterisation and enhanced pharmacokinetics of scFv-albumin fusions expressed in Saccharomyces cerevisiae.

Publication Date: 2010 Oct PMID: 20546898
Authors: Evans, L. - Hughes, M. - Waters, J. - Cameron, J. - Dodsworth, N. - Tooth, D. - Greenfield, A. - Sleep, D.
Journal: Protein Expr Purif

An expression system is described for the production of monomeric scFvs and scFv antibody fragments genetically fused to human albumin (at either the N- or C-terminus or both). Based upon strains of Saccharomyces cerevisiae originally developed for the production of a recombinant human albumin (Recombumin) this system has delivered high levels of secreted product into the supernatant of shake flask and high cell density fed-batch fermentations. Specific binding to the corresponding ligand was demonstrated for each of the scFvs and scFv-albumin fusions and pharmacokinetic studies showed that the fusion products had greatly extended circulatory half-lives. The system described provides an attractive alternative to other microbial systems for the manufacture of this type of product.

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Expression, purification and characterization of the cancer-germline antigen GAGE12I: a candidate for cancer immunotherapy.

Publication Date: 2010 Oct PMID: 20546897
Authors: Gjerstorff, M. F. - Besir, H. - Larsen, M. R. - Ditzel, H. J.
Journal: Protein Expr Purif

GAGE cancer-germline antigens are frequently expressed in a broad range of different cancers, while their expression in normal tissues is limited to the germ cells of the immune privileged organs, testis and ovary. GAGE proteins are immunogenic in humans, which make them promising targets for immunotherapy and candidates for cancer vaccines. Recombinant proteins may be superior to peptides as immunogens, since they have the potential to prime both CD4(+) and CD8(+) T cells and are not dependent on patient HLA-type. We have developed a method for production of highly pure recombinant GAGE12I-His by intracellular expression in yeast (Pichia pastoris) and nickel affinity, ion exchange and gel filtration purification. The identity of the purified protein was confirmed by mass spectrometry. This strategy yielded a total of 48 mg of highly pure (>98%) GAGE12I from 8 L of culture (6 mg/l). Interestingly, gel filtration and formaldehyde cross-linking indicated that GAGE12I forms tetramers. The purified recombinant GAGE12I represents a candidate molecule for vaccination of cancer patients and will form the basis for further structural analysis of GAGE proteins.

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High-level expression, purification and antibacterial activity of bovine lactoferricin and lactoferrampin in Photorhabdus luminescens.

Publication Date: 2010 Oct PMID: 20510367
Authors: Tang, Z. - Zhang, Y. - Stewart, A. F. - Geng, M. - Tang, X. - Tu, Q. - Yin, Y.
Journal: Protein Expr Purif

Bovine lactoferricin (LFC) and bovine lactoferrampin (LFA) are two active fragments located in the N(1)-domain of bovine lactoferrin. Recent studies suggested that LFC and LFA have broad-spectrum activity against Gram-positive and Gram-negative bacteria. To date, LFC and LFA have usually been produced from milk. We report here the high-level expression, purification and characterization of LFC and LFA using the Photorhabdus luminescens expression system. After the cipA and cipB genes were deleted by ET recombination, the expression host P. luminescens TZR(001) was constructed. A synthetic LFC-LFA gene containing LFC and LFA was fused with the cipB gene to form a cipB-LFC-LFA gene. To obtain the expression vector pBAD-cipB-LFC-LFA, the cipB-LFC-LFA gene was cloned on the L-arabinose-inducible expression vector pBAD24. pBAD-cipB-LFC-LFA was transformed into P. luminescens TZR(001). The cipB-LFC-LFA fusion protein was expressed under the induction of L-arabinose and its yield reached 12 mg L(-1) bacterial culture. Recombinant LFC-LFA was released from cipB by pepsin. The MIC of recombinant LFC-LFA toward E. coli 0149, 0141 and 020 was 6.25, 12.5 and 3.175 microg ml(-1), respectively.

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Of the vulnerability of orphan complex proteins: the case study of the E. coli IscU and IscS proteins.

Publication Date: 2010 Oct PMID: 20471481
Authors: Prischi, F. - Pastore, C. - Carroni, M. - Iannuzzi, C. - Adinolfi, S. - Temussi, P. - Pastore, A.
Journal: Protein Expr Purif

IscS and IscU, the two central protein components of the iron sulfur cluster assembly machinery, form a complex that is still relatively poorly characterized. In an attempt to standardize the purification of these proteins for structural studies we have developed a protocol to produce them individually in high concentration and purity. We show that IscS is a rather robust protein as long as it is produced in a PLP loaded form and that this co-factor is essential for fold stability and enzyme activity. In contrast to previous evidence, we also propose that, in contrast with previous evidence, IscU is a thermodynamically stable protein with a well defined fold but, when produced in isolation, is a 'complex-orphan protein' that is prone to unfolding if not stabilised by a co-factor or a protein partner. Our work will facilitate further structural and functional studies of these proteins and eventually lead to a better understanding of the whole machinery.

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Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes.

Publication Date: 2010 Oct PMID: 20460155
Authors: Kocabiyik, S. - Ozdemir, I. - Zwickl, P. - Ozdogan, S.
Journal: Protein Expr Purif

In this study we describe, the construction of a co-expression vector allowing simultaneous production of Thermoplasma volcanium 20S proteasome alpha- and beta-subunits in Escherichia coli. This heterologous expression system provided high level production of fully active 20S proteasome that can be purified easily by using a conventional two-step chromatographic technique. The recombinant proteasome was purified to homogeneity 12-fold with a specific activity of 26.5 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of two unique bands of alpha-(24 kDa) and beta-(21 kDa) subunits which were combined into proteolytically active proteasome complex in vivo when they were co-expressed in E. coli. The predominant peptide hydrolyzing activity was measured with typical chromogenic substrate (Ala-Ala-Phe-pNA) for chymotrypsin-like activity. The sequence analyses of the subunit genes showed that functional domains and residues including catalytic groups are highly conserved as compared to other archaeal proteasomes. Structural analysis by electron microscopy of negatively stained T. volcanium 20S proteasome revealed a unique conformational architecture (i.e. a tubular structure of four-stacked heptameric rings with a sevenfold symmetric top view) that is perfectly conserved from procaryotes to human.

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Human SUMO fusion systems enhance protein expression and solubility.

Publication Date: 2010 Oct PMID: 20457256
Authors: Wang, Z. - Li, H. - Guan, W. - Ling, H. - Wang, Z. - Mu, T. - Shuler, F. D. - Fang, X.
Journal: Protein Expr Purif

A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins.

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Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85alpha-p110alpha fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors.

Publication Date: 2010 Oct PMID: 20457255
Authors: Sinnamon, R. H. - McDevitt, P. - Pietrak, B. L. - Leydon, V. R. - Xue, Y. - Lehr, R. - Qi, H. - Burns, M. - Elkins, P. - Ward, P. - Vincentini, G. - Fisher, D. - Grimes, M. - Brandt, M. - Auger, K. R. - Ho, T. - Johanson, K. - Jones, C. S. - Schwartz, B. - Sweitzer, T. D. - Kirkpatrick, R. B.
Journal: Protein Expr Purif

Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.

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Escherichia coli expression, purification and characterization of functional full-length recombinant alpha2beta2gamma3 heterotrimeric complex of human AMP-activated protein kinase.

Publication Date: 2010 Oct PMID: 20451617
Authors: Rajamohan, F. - Harris, M. S. - Frisbie, R. K. - Hoth, L. R. - Geoghegan, K. F. - Valentine, J. J. - Reyes, A. R. - Landro, J. A. - Qiu, X. - Kurumbail, R. G.
Journal: Protein Expr Purif

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome. To date, expression of recombinant full-length human AMPK alpha2beta2gamma3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK alpha2beta2gamma3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK alpha2beta2gamma3 were transcribed as a single tricistronic transcript driven by the T7 RNA polymerase promoter, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. The self-assembled trimeric complex was purified from the cell lysate by nickel-ion chromatography using the hexahistidine tag fused exclusively at the N-terminus of the alpha 2 domain. The un-assembled beta 2 and gamma 3 domains were removed by extensive washing of the column. Further purification of the heterotrimer was performed using size exclusion chromatography. The final yield of the recombinant AMPK alpha2beta2gamma3 complex was 1.1mg/L culture in shaker flasks. The E. coli expressed enzyme was catalytically inactive after purification, but was activated in vitro by upstream kinases such as CaMKKbeta and LKB1. The kinase activity of activated AMPK alpha2beta2gamma3 complex was significantly enhanced by AMP (an allosteric activator) but not by thienopyridone A-769662, a known small molecule activator of AMPK. Mass spectrometric characterization of recombinant AMPK alpha2beta2gamma3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.

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A high-throughput purification of monoclonal antibodies from glycoengineered Pichia pastoris.

Publication Date: 2010 Nov PMID: 20447459
Authors: Jiang, Y. - Li, F. - Button, M. - Cukan, M. - Moore, R. - Sharkey, N. - Li, H.
Journal: Protein Expr Purif

Glycoengineered Pichia pastoris provides a unique platform for screening monoclonal antibody (mAb) leads and high expressing strains. A simple, economic, and high-throughput purification for mAb from P. pastoris fermentation has been developed that can be easily operated in various commercially available liquid handlers. The method includes the use of STREAMLINE rProtein A in a 96-well platform and demonstrates good linear alignment and reproducibility in a wide concentration range. The antibody titers measured by the method have less than 15% variation in comparison to spiking titers. The mAb titer and quality obtained from this method are comparable to that from conventional column chromatography. The method can process hundreds of expression screening samples in a day, not only to accurately determine titers, but also to generate milligram quantities of mAb for quality assessment, including purity, folding, glycosylation, and antigen binding affinity.

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Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.).

Publication Date: 2010 Nov PMID: 20447458
Authors: Zhang, D. - Nandi, S. - Bryan, P. - Pettit, S. - Nguyen, D. - Santos, M. A. - Huang, N.
Journal: Protein Expr Purif

Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug's therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood-borne pathogens from the use of human or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for native transferrin. We expressed recombinant human transferrin in rice (Oryza sativa L.) at a high level of 1% seed dry weight (10 g/kg). The recombinant human transferrin was able to be extracted with saline buffers and then purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived recombinant human transferrin was shown to be not only structurally similar to the native human transferrin, but also functionally the same as native transferrin in terms of reversible iron binding and promoting cell growth and productivity. These results indicate that rice-derived recombinant human transferrin should be a safe and low cost alternative to human or animal plasma-derived transferrin for use in cell culture-based biopharmaceutical production of protein therapeutics and vaccines.

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Overexpression and purification of PWL2D, a mutant of the effector protein PWL2 from Magnaporthe grisea.

Publication Date: 2010 Nov PMID: 20438845
Authors: Schneider, D. R. - Saraiva, A. M. - Azzoni, A. R. - Miranda, H. R. - de Toledo, M. A. - Pelloso, A. C. - Souza, A. P.
Journal: Protein Expr Purif

The rice blast disease caused by the ascomycete Magnaporthe grisea continues to cause a tremendous impact in rice (Oryza sativa) cultures around the world. Elucidating the molecular basis of the fungus interactions with its host might help increase the general understanding of the pathogen-host relationship. At the moment of invasion, the fungus secretes effectors that modify host defenses and cellular processes as they successively invade living rice cells. PWL2, an effector protein, is a known AVR (avirulence) gene product. The PWL2 gene prevents the fungus from infecting weeping lovegrass (Eragrostis curvula). In this study, we identified a PWL2 allele gene (which we termed PWL2D) in a strain of M. grisea. The sequence of PWL2D has only two bases different from that of PWL2, producing alterations in residue 90 and residue 142. However, the alteration of residue 90 (from D(90) to N(90)) is critical to gene function. Here, we cloned the gene PWL2D in a pET System vector, expressed the gene product in Escherichia coli and evaluated by spectroscopic techniques some aspects of the PWL2D structure. While TRX-tagged PWL2D is prone to aggregation, the solubility of PWL2D is improved when it is overexpressed without its original signal peptide. Expression and purification procedures for these constructs are described. Finally, we found out that the protein seems to be an intrinsically disordered protein. Results from these studies will facilitate structural analysis of PWL2D and might contribute to understanding the gene's function and of fungal/plant interactions.

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Purification and characterization of Yersinia enterocolitica and Yersinia pestis LcrV-cholera toxin A(2)/B chimeras.

Publication Date: 2010 Nov PMID: 20438844
Authors: Tinker, J. K. - Davis, C. T. - Arlian, B. M.
Journal: Protein Expr Purif

Yersinia pestis is a virulent human pathogen and potential biological weapon. Despite a long history of research on this organism, there is no licensed vaccine to protect against pneumonic forms of Y. pestis disease. In the present study, plasmids were constructed to express cholera toxin A(2)/B chimeric molecules containing the LcrV protective antigen from Yersinia enterocolitica and Y. pestis. These chimeras were expressed and purified to high yields from the supernatant of transformed Escherichia coli. Western and GM(1) ELISA assays were used to characterize the composition, receptor-binding and relative stability of the LcrV-CTA(2)/B chimera in comparison to cholera toxin. In addition, we investigated the ability of the Y. pestis LcrV-CTA(2)/B chimera to bind to and internalize into cultured epithelial cells and macrophages by confocal microscopy. These studies indicate that the uptake and trafficking of the LcrV antigen from the chimera is comparable to the trafficking of native toxin. Together these findings report that stable, receptor-binding, non-toxic LcrV-cholera toxin A(2)/B chimeras can be expressed at high levels in E. coli and purified from the supernatant. In addition, the internalization of antigen in vitro reported here supports the development of these molecules as novel mucosal vaccine candidates.

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A purified C-terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant.

Publication Date: 2010 Nov PMID: 20438843
Authors: Singh, S. - Hedley, D. - Kara, E. - Gras, A. - Iwata, S. - Ruprecht, J. - Strange, P. G. - Byrne, B.
Journal: Protein Expr Purif

Recent high resolution structures of modified G-protein coupled receptors (GPCRs) have provided major insight into the mechanisms of receptor-ligand binding. However understanding of the complete mechanism of GPCR function remains limited. This study characterised C-terminally truncated versions of the human adenosine A(2A) receptor (A(2A)R) with a view to producing protein suitable for structural studies. The constructs terminated at residue A316, removing the intracellular C-terminal tail, or V334, producing a C-terminal tail equivalent in length to that of rhodopsin. Higher levels of functional receptor before and after solubilisation were obtained for both C-terminally truncated constructs compared to the wild-type receptor (WT) as assessed by radioligand binding analysis using [(3)H]ZM241385. The construct which yielded the highest level of functional receptor, V334 A(2A)R, was purified in DDM to high homogeneity with a final yield of 2 mg/L. Binding analysis revealed that the purified receptor had a specific activity of 20.2+/-1.2 nmol/mg, close to the theoretical maximum. Pure V334 A(2A)R was resistant to degradation over 15 days when stored at 4 degrees C or 20 degrees C and showed remarkable functional stability when stored at 4 degrees C, retaining 84% of initial functionality after 30 days. This construct is an excellent candidate for structural studies.

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Expression, purification and characterization of in vivo biotinylated dengue virus envelope domain III based tetravalent antigen.

Publication Date: 2010 Nov PMID: 20435144
Authors: Batra, G. - Talha, S. M. - Nemani, S. K. - Dhar, N. - Swaminathan, S. - Khanna, N.
Journal: Protein Expr Purif

Dengue is a rapidly spreading mosquito-borne viral disease prevalent in over a hundred countries around the world. A definitive identification of dengue infection depends on reliable dengue diagnostic tests. This study describes the design, expression and purification of an in vivo biotinylated chimeric dengue antigen to exploit the high affinity of biotin-streptavidin interaction to detect anti-dengue antibodies. This chimeric antigen incorporates the envelope domain III (EDIII) of the four dengue virus serotypes. A biotin acceptor peptide was fused with the chimeric dengue antigen for in vivo biotinylation in Escherichia coli through simultaneous co-expression of the biotin ligase, BirA. Despite the localization of the chimeric dengue antigen to the insoluble fraction of induced E. coli cells, it was found to be biotinylated in vivo. It was purified to near homogeneity using affinity chromatography with final yields of 20mg protein of approximately 95% purity, from 1L of induced E. coli shake flask culture, and the efficiency of biotinylation was estimated to be approximately 85%. Mouse antibodies specific to recombinant EDIII of each of the four dengue serotypes, captured on microtiter wells sensitized with anti-mouse immunoglobulin antibodies, were recognized specifically and with high efficiency by the biotinylated antigen in conjunction with streptavidin-enzyme conjugate. An evaluation of the biotinylated antigen against a panel of pre-characterized dengue-positive and dengue-negative human sera (n=164), in an antibody capture ELISA format, showed that it manifested 100% specificity, but also suggested that additional epitopes may need to be included in its design to enhance sensitivity.

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A selectively terminable transgenic rice line expressing human lactoferrin.

Publication Date: 2010 Nov PMID: 20433928
Authors: Lin, C. - Nie, P. - Lu, W. - Zhang, Q. - Li, J. - Shen, Z.
Journal: Protein Expr Purif

Human lactoferrin (hLF) is a multifunctional milk protein which could be utilized for promoting human health. Transgenic rice has been used as a bioreactor for mass production of recombinant hLF. However, one major concern over such transgenic rice is the risk of its unintended spreading into environment and into our food supplies. Here we report the development of selectively terminable transgenic rice expressing human lactoferrin in seeds. These transgenic rice plants could be selectively terminated by bentazon, a common herbicide used for rice weed control. The hLF expression cassette was constructed into a T-DNA containing the RNA interference cassette suppressing the expression of the rice gene CYP81A6 which detoxifies herbicide bentazon, and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers to glyphosate tolerance. A transgenic line, named as G281, was identified for its high sensitivity to bentazon, high tolerance to glyphosate, and high expression of hLF. Southern analysis suggested G281 is a single copy insertion event. Field tests demonstrated that G281 plants can be completely killed by a single spray of bentazon at 1000 mg/L, which is safe to regular rice and represents only half of the dose recommended by manufacturer for rice field weed control. Therefore, any G281 contaminations in regular rice could be selectively terminated to make sure it will not enter food or feed supplies.

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Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile.

Publication Date: 2010 Nov PMID: 20433927
Authors: Sundriyal, A. - Roberts, A. K. - Ling, R. - McGlashan, J. - Shone, C. C. - Acharya, K. R.
Journal: Protein Expr Purif

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.

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Expression, purification and characterization of the recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO), an active immunotherapeutic vaccine component.

Publication Date: 2010 Nov PMID: 20417283
Authors: Xu, B. - Lundgren, M. - Magnusson, A. C. - Fuentes, A.
Journal: Protein Expr Purif

The active vaccine component recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO) has been expressed in CHO-K1 cells. It contains two identical polypeptide chains with 338 amino acid residues in each chain connected by two disulfide bridges. The cell lines were adapted to suspension culture in a serum-free medium. An expression level of 60 mg/L was obtained after 8 days in a shaking flask at a temperature of 31.5 degrees C. The OSO protein has been purified to homogeneity by a combination of three chromatographic steps. Virus inactivation and reduction by solvent detergent treatment and nano-filtration were included in the process. The residual host cell protein content was less than 50 ng/mg OSO as analyzed by ELISA. Purity was analyzed by SDS-PAGE under reducing and non-reducing conditions and was estimated by densitometry to be above 99.0%. The dimer content was less than 0.1% as estimated by analytical size exclusion chromatography. The molecular mass, as estimated by SDS-PAGE, is 90 kDa. A value of around 74 kDa was calculated from its amino acid composition. This indicates that the protein is heavily glycosylated containing around 18% carbohydrate. Isoelectric focusing in polyacrylamide gel disclosed a ladder type band pattern with pI values in the pH-range 7.0-8.3, indicating a variation in the sialic acid content. The OSO protein is not stable at temperatures above 40 degrees C and at pH values below 4 indicating that virus inactivation by incubating the protein solution at higher temperature or at lower pH is not possible.

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Expression of Bv8 in Pichia pastoris to identify structural features for receptor binding.

Publication Date: 2010 Sep PMID: 20412858
Authors: Miele, R. - Lattanzi, R. - Bonaccorsi di Patti, M. C. - Paiardini, A. - Negri, L. - Barra, D.
Journal: Protein Expr Purif

Bv8 is an amphibian peptide belonging to the widely distributed AVIT protein family. The mammalian orthologues of Bv8 were named prokineticin 1 and prokineticin 2. Two G-protein-coupled receptors for Bv8-prokineticins have been identified. The biological activities of Bv8/PK proteins range from angiogenesis and involvement in reproduction and cancer, to neuronal survival and neurogenesis, hypothalamic hormone secretion, circadian rhythm control and immunomodulatory processes. Identifying the structural determinants required for receptor binding of Bv8-PKs is mandatory for the design of PKR antagonists, which may be useful in the treatment and prevention of various disease states. Here we describe a procedure for the production in Pichia pastoris of Bv8 and 3 mutants: W24A-Bv8, in which the tryptophan in position 24 is substituted by alanine, the double mutant M1-W24A-Bv8, that contains an additional methionine at the N-terminus and Bv8-TyrTyr that includes two additional tyrosines at the C-terminus. The results evidence a relevant role of tryptophan 24 in Bv8-PKRs interaction.

MeSH Categories: Amphibian Proteins/*biosynthesis/chemistry/genetics/metabolism, Animals, Anura/*genetics, Electrophoresis, Polyacrylamide Gel, Kinetics, Models, Molecular, Mutation, Neuropeptides/*biosynthesis/chemistry/genetics/metabolism, Pichia/*genetics/metabolism, Protein Binding, Recombinant Proteins/chemistry/genetics/*metabolism, Tryptophan

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Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis.

Publication Date: 2010 Sep PMID: 20412857
Authors: Hamdane, D. - Skouloubris, S. - Myllykallio, H. - Golinelli-Pimpaneau, B.
Journal: Protein Expr Purif

Folate-dependent tRNA m(5)U methyltransferase TrmFO is a flavoprotein that catalyzes the C(5)-methylation of uridine at position 54 in the TPsiC loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)(6)-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein.

MeSH Categories: Bacillus subtilis/*enzymology/genetics, Bacterial Proteins/*biosynthesis/chemistry/genetics/isolation &, purification, Chromatography, Affinity/methods, Chromatography, Gel/methods, Circular Dichroism, Electrophoresis, Polyacrylamide Gel, Escherichia coli/genetics/metabolism, Histidine, Methylation, Models, Molecular, Molecular Weight, Mutation, NAD/metabolism, Oxidation-Reduction, Recombinant Fusion Proteins/*biosynthesis/chemistry/genetics/isolation &, purification, tRNA Methyltransferases/*biosynthesis/chemistry/genetics/isolation &, purification

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High-level expression of barley beta-D-glucan exohydrolase HvExoI from a codon-optimized cDNA in Pichia pastoris.

Publication Date: 2010 Sep PMID: 20406687
Authors: Luang, S. - Hrmova, M. - Ketudat Cairns, J. R.
Journal: Protein Expr Purif

The native beta-d-glucan exohydrolase isoenzyme ExoI from barley seedlings, designated HvExoI, was the first GH3 glycoside hydrolase, for which a crystal structure was determined. A precise understanding of relationships between structure and function in this enzyme has been gained by structural and enzymatic studies. To allow testing of hypotheses gained from these studies, an efficient system for expression of HvExoI in Pichia pastoris was developed using a codon-optimized cDNA. Protein expression at a temperature of 20 degrees C yielded a recombinant enzyme, designated rHvExoI, which had molecular masses of 70-110 kDa due to heavy glycosylation at Asn221, Asn498 and Asn600, the three sites of N-glycosylation in native HvExoI. Most of the N-linked carbohydrate could be removed from rHvExoI, resulting in N-deglycosylated rHvExoI with a substantially decreased molecular mass of 67 kDa. rHvExoI was able to hydrolyse barley (1,3;1,4)-beta-D-glucan, laminarin and lichenans. The catalytic efficiency value k(cat)/K(M) of rHvExoI with barley (1,3;1,4)-beta-D-glucan was similar to that reported for native HvExoI. Further, laminaribiose, cellobiose and gentiobiose were formed through transglycosylation reactions with 4-nitrophenyl beta-D-glucoside and barley (1,3;1,4)-beta-D-glucan. Overall, the biochemical properties of rHvExoI were similar to those reported for native HvExoI, although differences were seen in thermostabilities and hydrolytic rates of certain beta-linked glucosides.

MeSH Categories: Amino Acid Sequence, Chromatography, Thin Layer, Cloning, Molecular/*methods, Codon, DNA, Complementary, Electrophoresis, Polyacrylamide Gel, Glucosidases/*biosynthesis/chemistry/genetics, Glycosylation, Hordeum/*enzymology/genetics, Hydrogen-Ion Concentration, Kinetics, Molecular Sequence Data, Molecular Weight, Peptide Fragments, Pichia/*genetics/metabolism, Polysaccharides/metabolism, Recombinant Proteins/biosynthesis/chemistry/genetics, Substrate Specificity, Temperature, Trypsin/metabolism

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Characterization of mammalian equilibrative nucleoside transporters (ENTs) by mass spectrometry.

Publication Date: 2010 Sep PMID: 20399865
Authors: Reyes, G. - Naydenova, Z. - Abdulla, P. - Chalsev, M. - Villani, A. - Rose, J. B. - Chaudary, N. - DeSouza, L. - Siu, K. W. - Coe, I. R.
Journal: Protein Expr Purif

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.

MeSH Categories: Animals, COS Cells, Cercopithecus aethiops, Equilibrative Nucleoside Transport Proteins/*chemistry/genetics/metabolism, Mice, Peptide Fragments/chemistry/metabolism, Peptides/genetics/metabolism, Recombinant Fusion Proteins/chemistry/genetics/metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods, Thioinosine/analogs & derivatives/metabolism, Trypsin

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Functional expression, purification and high sequence coverage mass spectrometric characterization of human excitatory amino acid transporter EAAT2.

Publication Date: 2010 Nov PMID: 20399272
Authors: Ye, R. - Rhoderick, J. F. - Thompson, C. M. - Bridges, R. J.
Journal: Protein Expr Purif

The glial excitatory amino acid transporter 2 (EAAT2) mediates a majority of glutamate re-uptake in human CNS and, consequently, is associated with a variety of signaling and pathological processes. While our understanding of the function, mechanism and structure of this integral membrane protein is increasing, little if any mass spectrometric (MS) data is available for any of the EAATs specifically, and for only a few mammalian plasma membrane transporters in general. A protocol to express and purify functional EAAT2 in sufficient quantities to carry out MS-based peptide mapping as needed to study ligand-transporter interactions is described. A 6xHIS epitope was incorporated into the N-terminus of human EAAT2. The recombinant protein was expressed in high levels in mammalian HEK 293T cells, where it exhibited the pharmacological properties of the native transporter. EAAT2 was purified from isolated cell membranes in a single step using nickel affinity chromatography. In-gel and in-solution trypsin digestions were conducted on the isolated protein and then analyzed by MALDI-TOF and LC-MS/MS mass spectrometry. Overall, 89% sequence coverage of the protein was achieved with these methods. In particular, an 88 amino acid tryptic peptide covering the presumed substrate binding domains HP1, TMD7, HP2, and TMD8 domains of EAAT2 was also identified after N-deglycosylation. Beyond the specific applicability to EAAT2, this study provides an efficient, simple and scalable approach to express, purify, digest and characterize integral membrane transporter proteins by mass spectrometry.

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Production and characterization of recombinant protein preparations of Endonuclease G-homologs from yeast, C. elegans and humans.

Publication Date: 2010 Sep PMID: 20382228
Authors: Kieper, J. - Lauber, C. - Gimadutdinow, O. - Urbanska, A. - Cymerman, I. - Ghosh, M. - Szczesny, B. - Meiss, G.
Journal: Protein Expr Purif

Nuc1p, CPS-6, EndoG and EXOG are evolutionary conserved mitochondrial nucleases from yeast, Caenorhabditis elegans and humans, respectively. These enzymes play an important role in programmed cell death as well as mitochondrial DNA-repair and recombination. Whereas a significant interest has been given to the cell biology of these proteins, in particular their recruitment during caspase-independent apoptosis, determination of their biochemical properties has lagged behind. In part, biochemical as well as structural analysis of mitochondrial nucleases has been hampered by the fact that upon cloning and overexpression in Escherichia coli these enzymes can exert considerable toxicity and tend to aggregate and form inclusion bodies. We have, therefore, established a uniform E. coli expression system allowing us to obtain these four evolutionary related nucleases in active form from the soluble as well as insoluble fractions of E. coli cell lysates. Using preparations of recombinant Nuc1p, CPS-6, EndoG and EXOG we have compared biochemical properties and the substrate specificities of these related nucleases on selected substrates in parallel. Whereas Nuc1p and EXOG in addition to their endonuclease activity exert 5'-3'-exonuclease activity, CPS-6 and EndoG predominantly are endonucleases. These findings allow speculating that the mechanisms of action of these related nucleases in cell death as well as DNA-repair and recombination differ according to their enzyme activities and substrate specificities.

MeSH Categories: Amino Acid Sequence, Animals, Caenorhabditis elegans/genetics, Caenorhabditis elegans, Proteins/biosynthesis/*chemistry/genetics/metabolism, DNA/chemistry/genetics/metabolism, DNA Repair, Endodeoxyribonucleases/biosynthesis/*chemistry/genetics/metabolism, Endonucleases/biosynthesis/*chemistry/genetics/metabolism, Escherichia coli/genetics/metabolism, Humans, Hydrogen-Ion Concentration, Mitochondrial Proteins/biosynthesis/*chemistry/genetics/metabolism, Molecular Sequence Data, Recombinant Proteins/biosynthesis/chemistry/genetics, Saccharomyces cerevisiae/genetics, Saccharomyces cerevisiae Proteins/biosynthesis/chemistry/genetics, Sequence Alignment, Spectrometry, Fluorescence

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Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.

Publication Date: 2010 Sep PMID: 20381622
Authors: Tomala, M. - Lavrentieva, A. - Moretti, P. - Rinas, U. - Kasper, C. - Stahl, F. - Schambach, A. - Warlich, E. - Martin, U. - Cantz, T. - Scheper, T.
Journal: Protein Expr Purif

Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin.

MeSH Categories: Animals, Antigens, CD15/metabolism, Base Sequence, Cell Line, Cell Proliferation/drug effects, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Embryonic Stem Cells, Endopeptidases/metabolism, Escherichia coli/*genetics/metabolism, Flow Cytometry, Humans, Leukemia Inhibitory Factor/genetics/*isolation &, purification/metabolism/*pharmacology, Mice, Molecular Sequence Data, Recombinant Fusion Proteins/genetics/*isolation &, purification/metabolism/*pharmacology, Solubility, Thioredoxins/genetics/*metabolism

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Molecular cloning and expression in Pichia pastoris of a hypoallergenic antigen 5.

Publication Date: 2010 Sep PMID: 20371379
Authors: Vinzon, S. E. - Pirpignani, M. L. - Nowicki, C. - Biscoglio de Jimenez Bonino, M.
Journal: Protein Expr Purif

Stings by insects from the Hymenoptera order can cause life-threatening allergic reactions and impair life quality. Immunotherapy with venom extracts is the most extensively employed treatment to reduce morbidity and mortality, but purified and safer allergy vaccines are needed. Antigen 5 is an important allergen of vespid venoms. We previously reported that Antigen 5 from Polybia scutellaris (Poly s 5) is likely to be a hypoallergenic variant. On the basis of such findings, this work deals with the recombinant expression and purification of Poly s 5 in Pichia pastoris. In order to overcome non-native glycosylation of the recombinant protein, it was necessary to delete a glycosylation site. On the other hand, different strategies were attempted to obtain a satisfactory yield of the protein; moreover, the influence of the methanol concentration in the expression medium was investigated and found to be crucial. Mass spectrometry, N-terminal sequencing, and IgG-binding inhibition assays were performed. Results allowed us to confirm the immunological equivalence between the recombinant and the natural proteins. In conclusion, a novel protocol for the recombinant expression of Poly s 5 in P. pastoris was designed thus bringing about a high yield of the protein useful for clinical and scientific purposes.

MeSH Categories: Animals, Chromatography, Affinity, Cloning, Molecular/*methods, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Glycosylation, High-Throughput Screening Assays, Mice, Mutagenesis, Site-Directed, Pichia/*genetics/metabolism, Recombinant Proteins/biosynthesis/chemistry/genetics/immunology, Spectrometry, Mass, Electrospray Ionization, Wasp Venoms/*biosynthesis/chemistry/genetics/immunology

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High-efficient expression, refolding and purification of functional recombinant C-terminal fragment of human alpha-fetoprotein.

Publication Date: 2010 Sep PMID: 20363333
Authors: Sharapova, O. A. - Pozdnykova, N. V. - Laurinavichyute, D. K. - Yurkova, M. S. - Posypanova, G. A. - Fedorov, A. N. - Severin, S. E. - Severin, E. S.
Journal: Protein Expr Purif

Human alpha-fetoprotein (hAFP) is an oncofetal protein which is a common cancer marker. Conjugates of native hAFP with different cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The hAFP interacts with its receptor (AFPR) on the surface of cancer cells via its C-terminal domain. The aim of this work was to develop a highly efficient expression system in Escherichia coli and efficient refolding procedure for the recombinant C-terminal fragment of hAFP (rAFP-Cterm) and to characterize its functional properties. C-terminal fragment of hAFP (rAFP-Cterm) comprising amino acids from 404 to 609 was expressed in E. coli BL21 (DE3) strain with high yield. High efficient purification and refolding procedures were developed giving yield of refolded protein about 80% with purity about 95%. The refolded rAFP-Cterm bound specifically with cancer cells carrying AFPR and was accumulated by them with the same efficiency as native hAFP. This rAFP-Cterm can be used as a vehicle for the targeted delivery of drugs to cancer cells.

MeSH Categories: Cell Line, Tumor, Chromatography, Gel, Chromatography, Reverse-Phase, Circular Dichroism, Drug Delivery Systems, Humans, Peptide Fragments/*biosynthesis/chemistry/genetics/isolation &, purification, Protein Binding, Protein Folding, Recombinant Proteins/biosynthesis/chemistry/genetics, alpha-Fetoproteins/*biosynthesis/chemistry/genetics/isolation &, purification

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Purification and refolding of recombinant human interferon-gamma in urea-ammonium chloride solution.

Publication Date: 2010 Sep PMID: 20363329
Authors: Petrov, S. - Nacheva, G. - Ivanov, I.
Journal: Protein Expr Purif

A method for purification and refolding of recombinant human interferon-gamma (hIFNgamma) from inclusion bodies is described. It includes the following steps: (i) solubilization of inclusion bodies in 7.4 M guanidinium hydrochloride; (ii) purification of the denatured hIFNgamma by hydrophobic chromatography on Octyl-Sepharose column (one step elution with 6 M urea/1 M ammonium chloride); (iii) refolding of the partly purified protein in 0.75 M urea, 20 mM Tris-HCl, pH 8.2; (iv) purification of the refolded protein by CM-Sepharose chromatography. The protein thus obtained is characterized by the following general parameters: yield 1.0 mg/g wet cell mass; purity >99%; specific activity 2x10(8)IU/mg; stability - more than two years as a lyophilized powder and more than two months in solution at 4 degrees C.

MeSH Categories: Ammonium Chloride/*chemistry, Cell Line, Chromatography, Agarose, Electrophoresis, Polyacrylamide Gel, Escherichia coli/genetics/metabolism, Humans, Inclusion Bodies/chemistry/metabolism, Interferon-gamma/*biosynthesis/chemistry/genetics, Protein Folding, Recombinant Proteins/biosynthesis/chemistry/genetics, Solubility, Urea/*chemistry

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Expression and purification of metabotropic glutamate receptor 7 peptides.

Publication Date: 2010 Sep PMID: 20363328
Authors: Isozumi, N. - Ohki, S.
Journal: Protein Expr Purif

Metabotropic glutamate receptors (mGluRs) influence a variety of second-messenger systems and ion channels. The C-terminal region of group III mGluRs interacts with the Ca(2+)-binding protein calmodulin (CaM). We intend to study the interaction between Ca(2+)/CaM and the CaM-binding motifs within mGluR(7), which is a group III mGluR. We established a recombinant protein expression and purification system for nuclear magnetic resonance (NMR) analysis of mGluR(7) peptides using Escherichia coli. Peptides of mGluR(7) conjugated to an affinity tag sequence were constructed, and protocols for expression and purification were optimized. To suppress non-specific enzymatic cleavage, the mGluR(7) fusion peptide was bound to Ca(2+)/CaM before enterokinase cleavage. This complex method for precise enzymatic reactions may be applicable for the recombinant preparation of a wide variety of peptides.

MeSH Categories: Amino Acid Sequence, Animals, Base Sequence, Calcium/metabolism, Calmodulin/metabolism, Electrophoresis, Polyacrylamide Gel, Escherichia coli/genetics, Molecular Sequence Data, Nitrogen Isotopes/chemistry/metabolism, Nuclear Magnetic Resonance, Biomolecular, Peptide Fragments/*biosynthesis/chemistry/genetics, Rats, Receptors, Metabotropic, Glutamate/*biosynthesis/chemistry/genetics/isolation & purification, Recombinant Fusion Proteins/biosynthesis/chemistry/genetics/isolation &, purification

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A histidine substitution confers metal binding affinity to a Schistosoma japonicum Glutathione S-transferase.

Publication Date: 2010 Sep PMID: 20347989
Authors: Han, Y. H. - Seo, H. A. - Kim, G. H. - Lee, C. K. - Kang, Y. K. - Ryu, K. H. - Chung, Y. J.
Journal: Protein Expr Purif

Glutathione S-transferases (GSTs) are multifunctional enzymes that are used as fusion tags on recombinant proteins in mammalian and Escherichia coli expression systems. We recently found that the Schistosoma japonicum GST (SjGST) displays weak Ni(2+) ion binding affinity. Glu26 and His79 were assumed to be its Ni(2+) binding sites based on the structure of the 26-kDa Clonorchis sinensis GST. To enhance SjGST Ni(2+) binding affinity, Glu26 was mutated to His. SjGST-E26H was expressed and purified at a high concentration of imidazole to a higher purity than wild type SjGST. In addition, human biotin protein ligase fused to SjGST-E26H was purified with a immobilized Ni affinity column.

MeSH Categories: Amino Acid Sequence, Amino Acid Substitution, Animals, Biotin/genetics, Chromatography, Affinity/*methods, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Escherichia coli/genetics, Glutathione Transferase/chemistry/genetics/*metabolism, Histidine/chemistry/genetics/*metabolism, Humans, Models, Molecular, Molecular Sequence Data, Nickel/*metabolism, Recombinant Fusion Proteins/chemistry/genetics/*metabolism, Schistosoma japonicum/*enzymology/genetics, Sequence Alignment, Structure-Activity Relationship

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Codon optimization for enhanced Escherichia coli expression of human S100A11 and S100A1 proteins.

Publication Date: 2010 Sep PMID: 20347987
Authors: Marlatt, N. M. - Spratt, D. E. - Shaw, G. S.
Journal: Protein Expr Purif

The cloning, expression and purification for the recombinant full-length human proteins S100A11 and human S100A1 is described. The genes were synthesized by overlapping complementary single-stranded oligonucleotides of various lengths. The coding sequence for both genes were codon optimized by selecting only the most preferential codons according to the Escherichia coli bias. In order to assemble the various oligonucleotides into the correct full-length genes, a unique one-step PCR procedure was implemented. The expression and purification procedures were also optimized for each protein. A single phenyl-Sepharose column was sufficient for the purification of human S100A11 whereas HiTrap Q anion exchange followed by phenyl-Sepharose columns were required for the purification of S100A1. By optimizing the S100A1 and S100A11 gene, expression and purification protocols, more than 45 and 150mg, respectively of the purified human proteins were obtained per litre of media. Protein identity was verified by both SDS-PAGE and mass spectrometry (MS) and further characterized by NMR spectroscopy. These results have established an efficient method for the expression and purification of large quantities of human S100A1 and S100A11 proteins for biophysical characterization.

MeSH Categories: Amino Acid Sequence, Base Sequence, Cloning, Molecular/methods, *Codon, Conserved Sequence, EF Hand Motifs, Electrophoresis, Polyacrylamide Gel, Escherichia coli/*genetics/metabolism, Humans, Mass Spectrometry, Molecular Sequence Data, Nitrogen Isotopes/metabolism, Nuclear Magnetic Resonance, Biomolecular, Protein Isoforms, S100 Proteins/*biosynthesis/chemistry/genetics, Sequence Alignment

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Control of translational initiation in the wheat-embryo cell-free protein expression system for producing homogenous products.

Publication Date: 2010 Sep PMID: 20304073
Authors: Ohta, T. - Matsuoka, H. - Nomura, Y. - Tozawa, Y.
Journal: Protein Expr Purif

Wheat-embryo cell-free protein expression system allows efficient production of a wide variety of proteins. Homogeneity of the end products is an important characteristic of an advanced cell-free system that will be used in a field of protein science such as structural biology. A translation enhancer such as the omega sequence derived from tobacco mosaic virus, that allows cap-independent translation of the mRNA in the cell-free system, is required for low-cost preparation of template mRNAs in the cell-free translation system. However, the use of translational enhancers often leads to unexpected byproducts. Several AUU codons in the omega sequence can potentially function as translation initiators. We confirmed that the in-frame AUU in the omega sequence functions as a non-canonical start codon and results in the extension of the N-terminus of the target protein in some cases. Investigation of the selectivity of non-canonical initiation codon under the control of omega sequence in the wheat-embryo cell-free system revealed that seven non-AUG codons, CUG, AUA, AUU, GUG, ACG, AUC, and UUG, are recognized as translation initiators. We found that the introduction of an in-frame stop codon just upstream of the target open reading frame is an efficient way to avoid unexpected byproducts. This minor but effective modification facilitates production of homogeneous proteins within the wheat-embryo cell-free protein expression system at the preparative scale.

MeSH Categories: Amino Acid Sequence, Base Sequence, Cell-Free System, Cloning, Molecular, Codon/*genetics, Electrophoresis, Polyacrylamide Gel, Glutathione Transferase/biosynthesis/genetics, Green Fluorescent Proteins/biosynthesis/genetics, Molecular Sequence Data, Oryza sativa, Plant Extracts/metabolism, Plant Proteins/biosynthesis/*genetics, Protein Biosynthesis/*genetics, Pyrophosphatases/biosynthesis/genetics, Recombinant Proteins/biosynthesis/genetics, Seeds/*chemistry, *Transcription Initiation Site, Triticum/*chemistry

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Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli.

Publication Date: 2010 Sep PMID: 20303407
Authors: Sudhamsu, J. - Kabir, M. - Airola, M. V. - Patel, B. A. - Yeh, S. R. - Rousseau, D. L. - Crane, B. R.
Journal: Protein Expr Purif

Over-expression of heme binding proteins in Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His-ligated hemes.

MeSH Categories: Bacterial Proteins/chemistry/genetics/metabolism, Carrier Proteins/chemistry/genetics/metabolism, Cytochrome P-450 Enzyme System/chemistry/genetics/metabolism, Electrophoresis, Polyacrylamide Gel, Escherichia coli/*genetics/metabolism, Ferrochelatase/*biosynthesis/chemistry/genetics/metabolism, Heme/*metabolism, Hemeproteins/chemistry/genetics/metabolism, Histidine/genetics/metabolism, Nitric Oxide Synthase/chemistry/genetics/metabolism, Recombinant Fusion Proteins/biosynthesis/chemistry/genetics/metabolism, Spectrum Analysis, Spectrum Analysis, Raman

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A novel TWO-STEP renaturation procedure for efficient production of recombinant BMP-2.

Publication Date: 2010 Sep PMID: 20302941
Authors: von Einem, S. - Schwarz, E. - Rudolph, R.
Journal: Protein Expr Purif

Bone morphogenetic proteins (BMPs) stimulate bone formation and thus constitute important protein therapeutics. Here, a novel procedure is presented which allows fast and efficient production of biologically active BMP-2 via a TWO-STEP procedure: the conditions are designed such that the first step favors formation of monomeric species with the correct intramolecular disulfide bridges, the conditions of the second folding reaction stimulate the formation of the intermolecular disulfide bridge. The short processing times and increased yields compared to previously published procedures allow low-cost production of this important protein drug.

MeSH Categories: Alkaline Phosphatase/biosynthesis, Bone Morphogenetic Protein 2/*biosynthesis/chemistry/genetics, Electrophoresis, Polyacrylamide Gel, Enzyme Induction, Escherichia coli/genetics, Humans, Inclusion Bodies/chemistry/metabolism, Oxidation-Reduction, Protein Folding, Recombinant Proteins/biosynthesis/chemistry/genetics

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Expression, purification, cross-reactivity and homology modeling of peanut profilin.

Publication Date: 2010 Sep PMID: 20230899
Authors: Cabanos, C. - Tandang-Silvas, M. R. - Odijk, V. - Brostedt, P. - Tanaka, A. - Utsumi, S. - Maruyama, N.
Journal: Protein Expr Purif

Plant profilins are known pan-allergens involved in the cross-reactions between pollen and plant foods. Peanut profilin, Ara h 5, is one of the important peanut allergens. Presently, most immunological, biochemical and structural studies on peanut allergens have focused on the three major allergens (Ara h 1, 2 and 3). Here Ara h 5 was cloned, expressed in Escherichia coli, Rosetta2(DE3) (Novagen), purified using a combination of ammonium sulfate fractionation and size-exclusion chromatography and yielded a total of 29 mg/l of culture. IgE reactivity was assayed using multiplexed microarray with other peanut allergens (Ara h 1, 2, 3, and 8) and birch (Bet v 2) and timothy (Phl p 2) profilin using sera from peanut allergic Swedish patients. Using homology modeling, Ara h 5 structure was also generated, compared against other profilins and utilized to predict surface-exposed residues potentially forming epitopes. The allergen was recognized by 3 out of 33 sera (9.1%). IgE reactivity to Ara h 5 also coincided with that of two other profilins, Phl p 12 and Bet v 2, confirming cross-reactivity. Interestingly, IgE reactivity to Ara h 5 was higher than above-mentioned profilins which may be indicating specificity of sera towards peanut profilin. Eight surface-exposed epitopes were predicted and verified against experimentally validated sequential epitopes. Three epitopes (#1, 5 and 7) mostly located at the accessible loops and neutral to relatively electropositive sites were found common among profilins, which should be involved in cross-reactivity. A specific putative epitope (#4) was also found which may explain the relative high IgE reactivity to Ara h 5 as compared to the other profilins. Due to its close relation to other allergenic profilins, Ara h 5 could be used as a model and allergen of choice for profilin allergy diagnosis.

MeSH Categories: Allergens/chemistry/genetics/immunology/*metabolism, Amino Acid Sequence, Arachis hypogaea/immunology/*metabolism, Chemical Precipitation, Chromatography, Gel, Cloning, Molecular, Cross Reactions, Epitopes, B-Lymphocyte/chemistry, Escherichia coli/genetics, Humans, Immunoglobulin E/metabolism, Models, Molecular, Molecular Sequence Data, Plant Proteins/chemistry/genetics/immunology/*metabolism, Profilins/chemistry/genetics/immunology/*metabolism, Protein Array Analysis, Recombinant Proteins/chemistry/genetics/immunology/*metabolism, Sequence Alignment

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