Purification and characterization of a recombinant Yersinia pestis V-F1 “Reversed” fusion protein for use as a new subunit vaccine against plague
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- 作者：Jeremy L. Goodin ; Bradford S. Powell ; Jeff T. Enama ; Ronald W. Raab ; Robert L. McKown ; George L. Coffman ; Gerard P. Andrews
- 关键词：Yersinia pestis ; Vaccine ; F1-V ; F1S-V-F1 ; caf1
- 刊名：Protein Expression and Purification
- 出版年：2011
- 出版时间：March 2011
- 年：2011
- 卷：76
- 期：1
- 页码：136-144
- 全文大小：760 K
文摘
We previously developed a unique recombinant protein vaccine against plague composed of a fusion between the Fraction 1 capsular antigen (F1) and the V antigen. To determine if overall expression, solubility, and recovery of the F1-V fusion protein could be enhanced, we modified the original fusion. Standard recombinant DNA techniques were used to reverse the gene order such that the V antigen coding sequence was fused at its C-terminus to the N-terminus of F1. The F1 secretion signal sequence (F1S) was subsequently fused to the N-terminus of V. This new fusion protein, designated F1S-V-F1, was then co-expressed with the Y. pestis Caf1M periplasmic chaperone protein in BL21-Star Escherichia coli. Recombinant strains expressing F1-V, F1S-F1-V, or F1S-V-F1 were compared by cell fractionation, SDS–PAGE, Western blotting, and suspension immunolabelling. F1S-V-F1 exhibited enhanced solubility and secretion when co-expressed with Caf1M resulting in a recombinant protein that is processed in a similar manner to the native F1 protein. Purification of F1S-V-F1 was accomplished by anion-exchange and hydrophobic interaction chromatography. The purification method produced greater than 1 mg of purified soluble protein per liter of induced culture. F1S-V-F1 polymerization characteristics were comparable to the native F1. The purified F1S-V-F1 protein appeared equivalent to F1-V in its ability to be recognized by neutralizing antibodies.