1. Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge
- Author
-
Yonghai Yang, Baizhong Cui, Wang Wang, Tao-Xing Shi, Hu Wang, Zhizhen Qi, Xiaoyi Wang, Ziwen Zhu, Ruifu Yang, Benchuan Wu, Qingwen Zhang, Zuyun Wang, Zhaobiao Guo, Yefeng Qiu, and Ruixia Dai
- Subjects
Enteropeptidase ,Pore Forming Cytotoxic Proteins ,Yersinia pestis ,Health, Toxicology and Mutagenesis ,Protein subunit ,Recombinant Fusion Proteins ,Blotting, Western ,Genetic Vectors ,Molecular Sequence Data ,Protein Engineering ,law.invention ,Fusion gene ,Mice ,Thrombin ,Affinity chromatography ,law ,medicine ,Escherichia coli ,Animals ,LcrV ,Amino Acid Sequence ,Cloning, Molecular ,Antigens, Bacterial ,Mice, Inbred BALB C ,Plague ,Plague Vaccine ,biology ,Chemistry ,Public Health, Environmental and Occupational Health ,biology.organism_classification ,Virology ,Molecular biology ,Antibodies, Bacterial ,Survival Analysis ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Vaccines, Subunit ,Recombinant DNA ,Electrophoresis, Polyacrylamide Gel ,Female ,medicine.drug ,Plasmids - Abstract
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 Co2+ 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. Conclusion 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.
- Published
- 2010