Prokaryotic expression and purification of protease of HIV-1 HXB2 subtype and analysis of cleaving substrate protein Gag CAP2NC peptide
10.3724/SP.J.1008.2008.00365
- Author:
Ming CHEN
1
Author Information
1. Department of Microbiology
- Publication Type:Journal Article
- Keywords:
Cleave;
Gene expression;
HIV;
Protease
- From:
Academic Journal of Second Military Medical University
2010;29(4):365-370
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To express, purify and identify the protease (PR) of HIV-1 HXB2 subtype in E. coli, so as to screen the HIV-1 Gag CAP2/NC protein phage displayed library with randomized P2/NC protease cleavage sites and establish a phage model for in vitro screening of PR inhibitors. Methods: The primers were designed according to the PR amino acid sequence of HIV-1 HXB2 subtype and the E. coli preferred codon, the additional 5′-nucleotide sequence encoding the eight peptide MGTVS-FNF for autocleave sites was inserted into the upstream of PR sequence. Then the PR107 DNA sequence was cloned into pET-32a vector which was used as expression vector in E. coli. Expression of HIV PR was induced by IPTG in E. coli BL21-DE3 and the expressed PR protein was purified by the Ni-NTA affinity column. The purified PR protein was refolded by diluted with MES buffer and blended into substrate protein CAP2NC to test its cleaving activity and the result was identified by SDS-PAGE. Results: The HIV PR107 DNA fragment with E. coli preferred codon was synthesized and was successfully inserted into the expression vector pET-32a. HIV-1 PR was expressed in E. coli BL21 DE3 after the induction by IPTG with a relative molecular weight of 30 000. The purified PR protein has a concentration of 2.54 mg/ml, and after refolded it could cleave substrate protein CAP2NC and this effect can be blocked by PI agent. Conclusion: PR107 DNA fragment with E. coli preferred codon of HIV-1 HXB2 subtype has been successfully synthesized and the PR protein has been successfully expressed, which can cleave substrate protein CAP2NC.
