Design and expression recombinant protein of tcpA gene vibrio cholera in pET32a(+) vector

Vibrio cholerae is a gram-negative bacterial pathogen that causes diarrheal disease. One of the most important factors in the pathogenesis is Vibrio cholera. Pili also is co-regulated with toxin, which is necessary for bacterial colonization in intestine. The aim of this study was to examine recombinant protein expression and TcpA bioinformatics.

TcpA gene was studied for rare Codons existence and gene optimization conducted using bioinformatics software. Primers designed for amplification of synthetic tcpA gene. Synthetic gene was cloned in vector pET32a (). The recombinant plasmid pET32a ()-tcpA was transformed to E.coli strain BL21 (DE3). TcpA gene expression was induced by IPTG 1mM. Recombinant protein expression was evaluated using SDS PAGE and Western blotting. Ni-NTA affinity chromatography was used for purification of recombinant protein.

Codon adaptability index of naive tcpA gene was 0.6, while optimized gene had Codon adaptability index of 0.9. The prevalence ratio Codons increased to 75 percent through Codon optimization. Enzyme analysis approved tcpA gene cloning in pET32a () expression vector. A protein with a molecular weight of 38.6 kD was seen on SDS-PAGE and its reaction with the antihistidine antibodies was confirmed by Western blot. The purified protein amount was 11.533milligram per liter.

Optimization led to the expression of recombinant tcpA. Regarding tcpA antigenicity, this protein is a good candidate to develop immunity against cholera gene.