Efficient expression and purification of recombinant human m-calpain using an Escherichia coli expression system at low temperature.
Calpain belongs to the superfamily of Ca(2+)-regulated cysteine proteases, which are indispensable to the regulation of various cellular functions. Of the 15 mammalian calpain isoforms, µ- and m-calpains are the best characterized. Both µ- and m-calpain are ubiquitously expressed and exist as heterodimers, containing a distinct 80-kDa catalytic subunit (CAPN1 and CAPN2, respectively) and the common, 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for use in structural and physiological studies, however Escherichia coli systems have proven incompatible with large-scale preparation of calpain, with the exception of rat m-calpain. Here, we have established a highly efficient method to purify active recombinant human m-calpain using an E. coli expression system at low temperature (22°C). This was achieved by co-expressing CAPN2 with a C-terminal histidine-tag, and CAPNS1, lacking the first Gly-repeated region at the N-terminal. After three sequential passes through a chromatographic column, ~5 mg of human m-calpain was homogenously purified from 1 l of E. coli culture. Proteins were stable for several months. This is the first report of efficient, large-scale purification of recombinant human m-calpain using an E. coli expression system.
Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo 156-8506, Japan. hata-sj@igakuken.or.jp
Shoji
S
Hata
Mika
M
Ueno
Fujiko
F
Kitamura
Hiroyuki
H
Sorimachi
D002118:Calcium / Q000737:chemistry; D002154:Calpain / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D011994:Recombinant Proteins; D002154:Calpain; C449000:m-calpain; D002118:Calcium