RECONSTITUTION OF RABBIT SKELETAL-MUSCLE TROPONIN FROM THE RECOMBINANT SUBUNITS ALL EXPRESSED IN AND PURIFIED FROM ESCHERICHIA-COLI

Three subunits of rabbit skeletal muscle troponin were expressed in an d purified from Escherichia coli. The procedures were optimized, and t he reconstituted troponin complex is highly homogeneous, stable, and o btainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and puri fied are beta-TnT(N'-208), TnI(C64A, C133S), and the wild type TnC. Be ta-TnT(N'-208) is a 25 kDa fragment of beta-troponin T, which consists of 208 amino acids and lacks 58 residues in the N-terminal variable r egion. TnI(C64A, C133S) is a mutant troponin I, in which Cys-64 and Cy s-133 are replaced by Ala and Ser, respectively. Each subunit was sepa rately expressed in E. coli, purified by column chromatography includi ng HPLC, and reassembled to form troponin complex. The reconstituted t roponin complex was not distinguishable from authentic troponin prepar ed from rabbit skeletal muscle; the acto-Sl ATPase rate, as well as th e superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystalliz ation trials.