Molecular cloning of Clostridium perfringens epsilon-toxin gene and its high level expression in E. coli.
A gene coding for epsilon-toxin was isolated from a field isolate of Clostridium perfringens type D by PCR amplification and was cloned under the control of T5 promoter fused with six-histidine tag at the amino terminal end. Escherichia coli cells harbouring this construct expressed high levels of the recombinant protein in the form of inclusion bodies. The protein was purified using single step affinity chromatography on a Ni(2+)-nitrilotriacetic acid (NTA) agarose column. Upon immunization of rabbit with the purified recombinant protein, high antibody titre was detected. The antibodies raised against the recombinant protein were able to recognize the recombinant as well as the native toxin. Anti epsilon-toxin monoclonal antibody was able to detect the recombinant protein in a Western blot. N-terminal sequence of the recombinant protein matched with the known sequence of the toxin. At the shake flask level, up to 20 mg of pure epsilon-prototoxin was produced per litre of culture.
Goswami
P P
PP
Molecular Pathology Laboratory, Indian Veterinary Research Institute, Izatnagar, India.
Rupa
P
P
Prihar
N S
NS
Garg
L C
LC
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D000917:Antibody Formation; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D001428:Bacterial Vaccines; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000235:genetics* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D006639:Histidine; D011401:Promoter Regions, Genetic; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D014614:Vaccines, Synthetic
D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; C039811:Clostridium perfringens epsilon-toxin; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D006639:Histidine