Purification and characterization of a recombinant Yersinia pestis V-F1 "Reversed" fusion protein for use as a new subunit vaccine against plague.
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 1mg 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.
Bacteriology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA. jeremy.goodin@amedd.army.mil
Jeremy L
JL
Goodin
Bradford S
BS
Powell
Jeff T
JT
Enama
Ronald W
RW
Raab
Robert L
RL
McKown
George L
GL
Coffman
Gerard P
GP
Andrews
D057134:Antibodies, Neutralizing / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D010930:Plague / Q000517:prevention & control*; D010931:Plague Vaccine / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D022223:Vaccines, Subunit / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D015010:Yersinia pestis / Q000276:immunology*
D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; C043908:LcrV protein, Yersinia; D010931:Plague Vaccine; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D022223:Vaccines, Subunit