Expression of regulated cardiac troponin I in Escherichia coli.
The study of the functional effects of troponin isoform changes would be greatly aided by the development of a strategy permitting protein engineering and mutational analysis. To assess the role of troponin isoforms in regulating myofibrillar ATPase activity, we have expressed rat cardiac troponin I (cTnI) in E. coli and purified the protein to near homogeneity. We utilized the inducible expression vector pGEX-KG to create a glutathione-S-transferase fusion protein which can be cleaved with thrombin. Approximately 6 mg of cTnI can be purified from 1 l of culture. Ca2+Mg2+ ATPase activity was measured using the bacterially synthesized cTnI and the remaining components of the regulated actomyosin complex (troponin T, troponin C, tropomyosin, actin, and myosin) purified to homogeneity from mammalian hearts. In the presence of free Ca2+ ranging from 10(-2) to 10(-8) M, bacterially synthesized cTnI exhibits specific activity similar to that observed for control cTnI isolated from rat hearts. The bacterially synthesized protein is capable of stoichiometric phosphorylation and demonstrates appropriately regulated specific activity. These results establish the feasibility of using bacterial expression to study functional consequences of changes in expression of troponin isoforms.
Straceski
A J
AJ
Department of Medicine, Montefiore Medical Center, Bronx, NY 10467.
Nakouzi
A S
AS
Malhotra
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis; D014336:Troponin / Q000096:biosynthesis / Q000235:genetics*; D019210:Troponin I
D011994:Recombinant Proteins; D014336:Troponin; D019210:Troponin I; D000251:Adenosine Triphosphatases