Polymerase chain reaction (PCR) was used to generate DNA encoding a 60 kDa stress protein of using primers complementary to sequences at the 5′ and 3′ ends of 60 kDa stress protein genes (encoding the ‘65 kDa antigens’) of and The predicted PCR product of 1.8 kb contained the entire coding sequence of an 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 strain JM83. Transformed bacteria, induced by IPTG, expressed an 85 kDa fusion protein comprising glutathione -transferase (GST) and 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 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.


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