Thermal Unfolding of the Cold-acclimated Type of Carp Light Meromyosin Expressed by Recombinant DNA in Escherichia coli.

cDNA encoding fast skeletal muscle light meromyosin (LMM) predominantly expressed in carp acclimated to a cold temperature of 10 degrees C was inserted into an Escherichia coli expression vector pET-11a. The resulting plasmid pET10 produced non-fused carp 10 degrees C-type LMM, yielding 10% of the total proteins in E. coli. The 10 degrees C-type LMM was purified by altered dialyses against high-and low-ionic-strength buffers and ion-exchange chromatography. An apparent molecular mass of the purified LMM was about 74,000 on SDS-PAGE, which was slightly larger than that previously reported for LMM isolated from carp acclimated to 10 degrees C. Transition temperatures (Tm) were 30.2 and 34.9 degrees C for the present 10 degrees C-type LMM on DSC analysis. This LMM exhibited a typical pattern of alpha-helix in CD spectroscopy with two minima at 222 and 208 nm, and its alpha-helical content at 20 degrees C was about 70%. The maximal decreasing rate derivative at 35 degrees C of the mean residue ellipticity of carp LMM per unit change of measuring temperature well reflected Tm values observed in DSC analysis.