Bacterial Expression, Purification, and Characterization of Akazara Scallop Troponin C

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  • Ojima Takao
    Department of Chemistry, Faculty of Fisheries, Hokkaido University
  • Maita Michiko
    Department of Chemistry, Faculty of Fisheries, Hokkaido University
  • Inoue Akira
    Department of Chemistry, Faculty of Fisheries, Hokkaido University
  • Nishita Kiyoyoshi
    Department of Chemistry, Faculty of Fisheries, Hokkaido University

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タイトル別名
  • Bacterial Expression Purification and C

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To investigate structure-function relationship for akazara scallop troponin C (TnC), we constructed a bacterial expression system using a cDNA clone which we isolated previously (Ojima, T., Tanaka, H., and Nishita, K. Arch. Biochem. Biophys., 311, 272-276 (1994)). The cDNA for akazara scallop TnC was subcloned into the expression plasmid pET-16b and expressed in Escherichia coli BL21 (DE3) by the induction with isopropyl-β-D (-) -thiogalactopyranoside. Amount of the expressed TnC increased up to approximately 10% of total protein during 4h incubation. The TnC was thenextracted from the bacterial pellet and purified by subsequent column chromatographies on DEAE-Toyopearl 650M and Phenyl-Sepharose CL-4 B. Although the expressed TnC possessed an extra moiety of 6 amino acids at N-terminus, which was derived from adaptor DNA of λgt11 and 5'-untranslated region of the cDNA clone, it showed basically the same properties as those of native TnC with respect to the Ca2+-induced difference UV-absorption spectrum and Ca2+-regulatory ability when reconstitutedwith native tropo-nin T and troponin I.

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