Cloning and expression of a cholera toxin beta subunit in Escherichia coli

Abstract

Cholera is an acute infection of the intestine caused by theVibrio cholerae bacterium. This bacterium, a member of Vibrionaceae family, is a facultatively anaerobic, Gram-negative, non-spore-forming curved rod, about 1.4-2.6 μm long, and capable of both respiratory and fermentative metabolism. Cholera, characterized by numerous voluminous watery stools, is often accompanied by vomiting (leading to bicarbonate loss). One of the most important virulence factors of cholera is enterotoxin (ctxAB). The cholera toxin beta subunit (CTB) is a pentameric non-toxic portion of V. cholerae toxin, responsible for holotoxin binding to the GM1-ganglioside receptor that is present on most nucleated cells. When conjugated to autoantigens, CTB dramatically increases its tolerogenic potential after oral administration. In the present study, the CTB gene from a hypertoxigenic V. cholerae strain was amplified and cloned. We first amplified the CTB gene with specific primers and the Prime STAR enzyme. The amplified CTB gene was then cloned in pET-28 vector and introduced into Escherichia coli DH5α. pET plasmids containing the CTB gene plasmids were then extracted from the bacteria and used to transform an expression host (BL21). After culturing and induction of positive bacteria, SDS-PAGE and Western blotting for protein determination and verification were performed. We also quantified the level of recombinant CTB by ELISA. Our results show that, due to its rapid growth, an expression host such as E. coli can be useful for production of CTB protein. The ELISA results showed yields of recombinant protein of up to 1 mg/L culture. However, optimal conditions for expression in our study included choice of host strain, temperature used for culture, and concentration of antibiotic and inducer. Beta subunit has many important scientific applications. There is a great deal of interest in the use of CTB as an adjuvant for vaccines targeted for delivery to the mucosa-associated lymphatic tissues. The importance and multifunctional nature of Beta subunit of V. cholerae enterotoxin, e.g., in oral vaccine preparation, will make the use of prokaryotic systems (such as Escherichia coli) for production of this protein most advantageous. © Springer-Verlag and the University of Milan 2010.