Overexpression and characterization of human cardiac actin and mutants pertaining to cardiomyopathy.

摘要

The influence of the promoter and N-terminal hexahistidine tag on human cardiac actin (ACTC) expression and function was investigated using four baculovirus expression constructs. Both non-tagged and hexahistidine-tagged ACTC expression from the p10 promoter was higher than that from the polh promoter. Characterization showed that an N-terminal hexahistidine tag has a negative effect on ACTC function. Recombinant ACTC inhibits DNase-I and binds myosin S1, indicative of proper folding. The data support the hypothesis that the actin protein down-regulates the polh promoter. The ACTC mutant E99K, which has been associated with hypertrophic cardiomyopathy, was also expressed under the control of the p10 promoter and studies to investigate polymerization kinetics, filament and monomer stability and its interactions with myosin were completed. The E99K mutation resulted in weakened actomyosin interactions and compromised sarcomeric fiber force generation.