Abstract
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.
Highlights
From the Departments of Biochemistry and wiology, Queen's University, Kingston, Ontario, K7L 3N6 Canada cDNA for the C-terminal Ca2+-binding domain of rat pain proteins obtained from chicken, cow, and pig
The encodedprotein(21 m a ), Small subunit cDNA sequences have been reported from nuwhich correspondsclosely to the natural autolysisprod- merous species
Rat calpainI1 large subunit(80kDa) was produced from The process is laborious, involving four or five column steps, a cDNA clone in E. coli in soluble form at a level of and itis very difficult to eliminate all contamination with parapproximately 1 mgfliter
Summary
H grown to a n AsOOnmof 0.7 at 30 "C, and expression was induced by gtc agc gct aca gaa ctc atg aat atc ctc aac aag gtc gtg acc 135 addition of IPTG' to a final concentrationof 0.4 mM. PStI " " _ " " " " " ggc act gga cag atc caa gtg aac atcCAG GAG TGG D G CAG CTG 540 GTGQIQVNIQEWLQL mM Tris-HC1,pH 7.5, and assayed with casein at free Ccoan2c+entrations ranging from 0.05 to 8 mM. In these assays the final calpain concentrationwasapproximately0.1p~ (10 unitdassay);thefinal
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