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 subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled 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 phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.
Lohmann
K
K
Ruhr-Universität Bochum, Medizinische Fakultät, Institut für Physiologische Chemie, Abt. Biochemie Supramolekularer Systeme, 44780 Bochum, Germany.
Westerdorf
B
B
Maytum
R
R
Geeves
M A
MA
Jaquet
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008929:Mitochondria, Heart / Q000737:chemistry; D009206:Myocardium / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism; D014336:Troponin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases