Overproduction and purification of Mycobacterium tuberculosis chaperonin 10.
The overexpression of the Mycobacterium tuberculosis chaperonin 10 (Cpn10)-encoding gene was accomplished using baculovirus expression vectors. The product was immunoreactive with a Cpn10 monoclonal antibody (mAb) and had an electrophoretic mobility identical to authentic Cpn10. The baculovirus system was most successful in terms of reaching nearly the full expression potential of the system. Recombinant Cpn10 was purified from recombinant baculovirus-infected Spodoptera frugiperda cells by isoelectrofocussing and size-exclusion chromatography. The baculovirus vector and purification methodology described represent a very powerful system for the large-scale production of the M. tuberculosis Cpn10 which may allow us to undertake structure-function analysis.
Department of Medical Microbiology, St George's Hospital Medical School, London, UK.
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Atkins
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Prehaud
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Coates
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D018835:Chaperonin 10 / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011994:Recombinant Proteins; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D018835:Chaperonin 10; D017931:DNA Primers; D011994:Recombinant Proteins