Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge.
OBJECTIVE
LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study.


METHODS
A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography.


RESULTS
Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)₃ adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10⁶ CFU of Y. pestis virulent strain 141.


CONCLUSIONS
The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.
Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.
Wang
W
Wang
Zhi-Zhen
ZZ
Qi
Qing-Wen
QW
Zhang
Ben-Chuan
BC
Wu
Zi-Wen
ZW
Zhu
Yong-Hai
YH
Yang
Bai-Zhong
BZ
Cui
Rui-Xia
RX
Dai
Ye-Feng
YF
Qiu
Zu-Yun
ZY
Wang
Zhao-Biao
ZB
Guo
Tao-Xing
TX
Shi
Hu
H
Wang
Rui-Fu
RF
Yang
Xiao-Yi
XY
Wang
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010930:Plague / Q000276:immunology / Q000517:prevention & control*; D010931:Plague Vaccine / Q000235:genetics / Q000276:immunology*; D010957:Plasmids; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016019:Survival Analysis; D022223:Vaccines, Subunit / Q000235:genetics / Q000276:immunology; D015010:Yersinia pestis / Q000254:growth & development* / Q000276:immunology
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; C043908:LcrV protein, Yersinia; D010931:Plague Vaccine; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D022223:Vaccines, Subunit