Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain.

Fatemah Bakhshi,Bahram Golestani Imani,Reza Pilehchian Langroudi

doi:10.4102/ojvr.v83i1.1136

Abstract

Clostridium perfringens beta toxin is only produced by types B and C and plays an important role in many human and animal diseases, causing fatal conditions that originate in the intestines. We compared the expression of C. perfringens type B vaccine strain recombinant beta toxin gene in the Escherichia coli strains RosettaTM(DE3) and BL21(DE3). The beta toxin gene was extracted from pJETβ and ligated with pET22b(+). pET22β was transformed into E. coli strains BL21(DE3) and RosettaTM(DE3). Recombinant protein was expressed as a soluble protein after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction in strain RosettaTM(DE3) but not in BL21(DE3). Expression was optimised by growing recombinant cells at 37 °C and at an induction of 0.5 mM, 1 mM, 1.5 mM IPTG. Expression was evaluated using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified via Ni-NTA and was analysed using western blot. We concluded that E. coli strain RosettaTM(DE3) can enhance the expression of C. perfringens recombinant beta toxin.

Highlights

Clostridium perfringens, an anaerobic Gram-positive, rod-shaped bacterium, is able to form environmentally resistant spores
Expression of Clostridium perfringens beta toxin (CPB) in Escherichia coli has been shown and a molecular analysis revealed that it has sequence homology with alpha-toxin, gamma-toxin and leukocidin of Staphylococcus aureus (Hunter et al 1993)
Clostridium perfringens type B vaccine strain (CWB CN228), E. coli strain TOP10, which was applied as a cloning host, and E. coli strains BL21(DE3) and RosettaTM(DE3) (Novagen, Merck Millipore, Germany) as expression hosts were prepared at the Razi Institute

Summary

Introduction

Clostridium perfringens, an anaerobic Gram-positive, rod-shaped bacterium, is able to form environmentally resistant spores. Four of them – iota, alpha, beta and epsilon – are major toxins and are used for classifying C. perfringens into five types – A, B, C, D and E (Nilo 1980). Beta toxin is only produced by types B and C and plays an important role in many human and animal diseases via pore formation in the endothelial cell membrane (Michlard et al 2009; Nagahama et al 2015). Clostridium perfringens beta toxin (CPB) forms a multimeric transmembrane pore in the endothelial cell membrane and is the cause of cell lysis (Steinthorsdottir et al 2000). Clostridium perfringens beta toxin gene (cpb), which encodes a protein made up of 309 amino acids, is located on the different large plasmids that are carried by types B and C (Rokos, Rood & Duncan 1978). Expression of CPB in Escherichia coli has been shown and a molecular analysis revealed that it has sequence homology with alpha-toxin, gamma-toxin and leukocidin of Staphylococcus aureus (Hunter et al 1993)

Methods

Results

Conclusion