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) predominantl y expressed in carp acclimated to a cold temperature of 10 degrees C w as inserted into an Escherichia coli expression vector pET-11a. The re sulting plasmid pET10 produced non-fused carp 10 degrees C-type LMM, y ielding 10% of the total proteins in E. coli. The 10 degrees C-type LM M was purified by altered dialyses against high-and low-ionic-strength buffers and ion-exchange chromatography. An apparent molecular mass o f the purified LMM was about 74,000 on SDS-PAGE, which was slightly la rger than that previously reported for LMM isolated from carp acclimat ed to 10 degrees C. Transition temperatures (Tm) were 30.2 and 34.9 de grees C for the present 10 degrees C-type LMM on DSC analysis. This LM M exhibited a typical pattern of alpha-helix in CD spectroscopy with t wo minima at 222 and 208 nm, and its alpha-helical content at 20 degre es C was about 70%. The maximal decreasing rate derivative at 35 degre es C of the mean residue ellipticity of carp LMM per unit change of me asuring temperature well reflected Tm values observed in DSC analysis.