Active recombinant rat calpain II. Bacterially produced large and small subunits associate both in vivo and in vitro.
cDNA for the C-terminal Ca(2+)-binding domain of rat calpain small subunit was cloned by means of the polymerase chain reaction. The encoded protein (21 kDa), which corresponds closely to the natural autolysis product of the small subunit, was produced in soluble form in Escherichia coli at a level of 20 mg/liter of cell culture. Rat calpain II large subunit (80 kDa) was produced from a cDNA clone in E. coli in soluble form at a level of approximately 1 mg/liter. The 80-kDa subunit alone had no proteinase activity, with or without Ca2+, but Ca(2+)-dependent proteinase activity was obtained following association of the two subunits, which was achieved either by co-expression of the two subunit cDNAs in E. coli, or by mixing the two partially purified subunits in the presence of 1 M NaSCN followed by dialysis. The heterodimeric (80 + 21 kDa) proteinase had a Ca2+ requirement for 50% activity of 0.35 mM and a specific activity at 2 mM Ca2+ of approximately 1 unit/microgram, values essentially identical to those of natural (80 + 30 kDa) calpain II. The results establish association and biological activity of the bacterially produced subunits and provide a system for studying structure-function relationships in calpain by means of mutagenesis.
Graham-Siegenthaler
K
K
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Gauthier
S
S
Davies
P L
PL
Elce
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D002154:Calpain; D002118:Calcium