Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum

Goodin, Jeremy L.; Raab, Ronald W.; McKown, Robert L.; Coffman, George L.; Powell, Bradford S.; Enama, Jeffrey T.; Ligon, John A.; Andrews, Gerard P.

doi:10.1016/j.pep.2004.11.001

Cited by 14 publications

(10 citation statements)

References 27 publications

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“…For LC‐ESI/MS/MS, purified protein was digested with trypsin for peptide mapping and amino acid sequencing essentially as previously described (30) but with the following changes. The LC MSD Trap system (Agilent Technologies Inc., Wilmington, DE) was plumbed for capillary‐flow configuration, flow rate set to 20 μL/min, and 5 μL (4 pmol) of diluted sample was fractionated using a smaller Zorbax SB‐C8 column (1.0 mm × 150 mm, 3.5 μm) (Agilent).…”

Section: Methodsmentioning

confidence: 99%

Design and Testing for a Nontagged F1‐V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague

Powell

Andrews

Enama

et al. 2005

Biotechnology Progress

100

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A two-component recombinant fusion protein antigen was re-engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine-tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea-denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N-terminal methionine. Purity, quality, and higher-order structure were compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy, and multi-angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V protein also protected mice from wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1-V as the active pharmaceutical ingredient of the next plague vaccine.

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Section: Methodsmentioning

confidence: 99%

Design and Testing for a Nontagged F1‐V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague

Powell

Andrews

Enama

et al. 2005

Biotechnology Progress

100

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“…Proteins receiving the highest molecular weight search (MOWSE) scores were selected as the peptide fragments of AmyM (42). Three peptide fragments were detected, and the sequences of the three peptides were AESGLYAAIIGGK, GLLNDALTNNNYSR, and ASDGKPAGGIG WWASR, which shared 100% identity to the peptides from glucan-1,4-alpha-maltohexaohydrolase in the complete genome sequence of C. coralloides DSM 2259.…”

Section: Resultsmentioning

confidence: 99%

AmyM, a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. Strain EGB

Zhang

et al. 2015

Appl Environ Microbiol

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A novel ␣-amylase, AmyM, was purified from the culture supernatant of Corallococcus sp. strain EGB. AmyM is a maltohexaoseforming exoamylase with an apparent molecular mass of 43 kDa. Based on the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry and peptide mass fingerprinting of AmyM and by comparison to the genome sequence of Corallococcus coralloides DSM 2259, the AmyM gene was identified and cloned into Escherichia coli. amyM encodes a secretory amylase with a predicted signal peptide of 23 amino acid residues, which showed no significant identity with known and functionally verified amylases. amyM was expressed in E. coli BL21(DE3) cells with a hexahistidine tag. The signal peptide efficiently induced the secretion of mature AmyM in E. coli. Recombinant AmyM (rAmyM) was purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography, with a specific activity of up to 14,000 U/mg. rAmyM was optimally active at 50°C in Tris-HCl buffer (50 mM; pH 7.0) and stable at temperatures of <50°C. rAmyM was stable over a wide range of pH values (from pH 5.0 to 10.0) and highly tolerant to high concentrations of salts, detergents, and various organic solvents. Its activity toward starch was independent of calcium ions. The K m and V max of recombinant AmyM for soluble starch were 6.61 mg ml ؊1 and 44,301.5 mol min ؊1 mg ؊1 , respectively. End product analysis showed that maltohexaose accounted for 59.4% of the maltooligosaccharides produced. These characteristics indicate that AmyM has great potential in industrial applications.

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“…To test the expression of PsbO without thioredoxin, the PsbO cDNA was cloned into the pET28 system, which allowed expression of the recombinant protein lacking thioredoxin tag. In addition to the thioredoxin the pET32 system also includes a His 6 tag that in contrast to thioredoxin often decreases solubility of expressed proteins (Goodin et al 2005;Hamilton et al 2003;Routzahn and Waugh 2002). When using the pET32 vector, high-level expression of PsbO was induced and all of the recombinant protein was found in soluble (1) and PsbO isolated from spinach PSII (2).…”

Section: Discussionmentioning

confidence: 99%

Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli

et al. 2008

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PsbO protein is an important constituent of the water-oxidizing complex, located on the lumenal side of photosystem II. We report here the efficient expression of the spinach PsbO in E. coli where the solubility depends entirely on the formation of the disulfide bond. The PsbO protein purified from a pET32 system that includes thioredoxin fusion is properly folded and functionally active. Urea unfolding experiments imply that the reduction of the single disulfide bridge decreases stability of the protein. Analysis of inter-residue contact density through the PsbO molecule shows that Cys51 is located in a cluster with high contact density. Reduction of the Cys28-Cys51 bond is proposed to perturb the packing interactions in this cluster and destabilize the protein as a whole. Taken together, our results give evidence that PsbO exists in solution as a compact highly ordered structure, provided that the disulfide bridge is not reduced.

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Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum

Cited by 14 publications

References 27 publications

Design and Testing for a Nontagged F1‐V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague

Design and Testing for a Nontagged F1‐V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague

AmyM, a Novel Maltohexaose-Forming α-Amylase from Corallococcus sp. Strain EGB

Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli

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