Overexpression of human cardiac troponin in Escherichia coli: Its purification and characterization

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory s ubunit (cTnI), and the calcium binding subunit (cTnC), have been coexpresse d in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E, coli, The coexpressed subunits assembl ed within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC) . The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3: cTnI:cTnC was obtained. Upon pho sphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombina nt troponin complex shows a nearly identical P-31 NMR spectrum to the nativ e one isolated from bovine heart. By measuring the rate of myosin S1 bindin g to reconstituted thin filaments it was shown that the dependence of the r egulation of S1 binding upon calcium concentration and bisphosphorylation w as comparable to the native complex. (C) 2001 Academic Press.