Amplification, Cloning and Expression Assay of the minor subunit Colonization Factor Antigen I (CFaE) from Enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea in children under 5 years and it is also a cause of Travelers diarrhea worldwide. To prevent ETEC-caused diarrhea and decrease its prevalence، the WHO has promoted the vaccine production against this pathovar. CFA/I fimbriae is an important and frequent virulence factor of this bacteria، playing a critical role in pathogenesis. Therefore، tip protein of this fimbriae (CFaE) could describe as a vaccine candidate. This study was aimed at investigating the expression of the gene rCFaE from native sequence after cloning into expression vector. rCFaE was amplified by PCR using a newly designed set of primers. PCR product was then cloned into early cloning vector pTZ57R/T and then sub cloned into the expression vector pET28a (+). Protein expression in 2 strains of E. coli BL21 (DE3) pLysS and Rosetta was determined by using IPTG under different conditions. cloning and sub cloning process were performed successfully. Test samples in the comparatives with control samples did not show detectable protein on the SDS-PAGE. The same results were obtained after changing different parameters like time and temperature of induction and variety of IPTG concentration. It was not possible to obtain high level expression of the target recombinant protein production in E. coli with pattern codon usages، probably due to the high A+T content and also abundance rare codons in the native sequence of cfaE gene. Therefore we suggest synthesizing this gene after codon optimization and checking for expression that again.