Cloning and expression in Escherichia coli of DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis, the causative agent of Johne's disease.
Polymerase chain reaction (PCR) was used to generate DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis using primers complementary to sequences at the 5' and 3' ends of 60 kDa stress protein genes (encoding the '65 kDa antigens') of M. leprae and M. tuberculosis. The predicted PCR product of 1.8 kb contained the entire coding sequence of an M. paratuberculosis 60 kDa stress protein, with non-coding regions of 124 bp and 1 bp at the 5' and 3' ends, respectively. DNA encoding the entire ORF for the 60 kDa stress protein, as well as thrombin and Factor Xa proteolytic cleavage sites, was ligated into the bacterial expression vector pGEX-2T and used to transform Escherichia coli strain JM83. Transformed bacteria, induced by IPTG, expressed an 85 kDa fusion protein comprising glutathione S-transferase (GST) and M. paratuberculosis 60 kDa stress protein. This fusion protein was purified by adsorption to glutathione-agarose beads and shown to cross-react in Western blot analysis with an anti-mycobacterial 60 kDa stress protein monoclonal antibody. Recombinant M. paratuberculosis 60 kDa stress protein was liberated from GST by proteolytic cleavage with either thrombin or Factor Xa enzyme. Authenticity of liberated recombinant stress protein was confirmed by N-terminal amino acid sequencing.
Colston
A
A
Department of Veterinary Pathology, University of Edinburgh, Summerhall, UK.
McConnell
I
I
Bujdoso
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000472:pathogenicity; D010283:Paratuberculosis / Q000209:etiology; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015183:Restriction Mapping
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins