Construction and Expression of a Fused Gene for B Subunit of the Heat-Labile and a Truncated Form of the Heat-Stable Enterotoxins in Escherichia coli

Authors
Saeid Bouzari
Mana Oloomi
Ali Hatef Salmaninan
Anis Jafati
Abstract
Elaboration of different toxins by enterotoxigenic E. coli has been considered as one of the main virulence factors contributing to the manifestation of disease caused by these microorganisms. Various strategies have been employed to raise antibodies against these toxins as a line of defense. In this study, the 3’ terminus of the gene that codes for the binding subunit of the heat-labile enterotoxin of E. coli (LTB) was fused to the 5’ terminus of a truncated heat-stable enterotoxin of E. coli (ST) with a region coding for 7 amino acids separating the two moieties. The fused gene was sequenced and subsequently subcloned in the pET23a (+) expression vector in the EcoRI/HindIII sites. The construct was transferred into the E. coli strain, BL21 (DE3) pLysS and isopropyl-b -D-thiogalactopyranoside (IPTG) was used for induction. The expression of the LTB and ST in the fused protein was assessed using two commercially available kits. SDS-PAGE and Western blot were also used to examine the expressed protein. The results indicated the low expression of both toxin moieties that were recognizable by the commercially available antibodies and the expressed protein was non-toxic as indicated by suckling mouse assay.
Keywords
Heat-labile enterotoxin
Heat-Stable enterotoxin
E. coli