title: Efficient expression and purification of recombinant human μ-calpain using an Escherichia coli expression system.
abstract: Calpains comprise a superfamily of Ca(2+) -regulated cysteine proteases that are indispensable for the regulation of various cellular functions. Of these, the mammalian μ- and m-calpains are the best characterized isoforms. They are ubiquitously expressed and form heterodimers consisting of a distinct 80-kDa catalytic subunit (CAPN1 for μ-calpain and CAPN2 for m-calpain) and a common 30-kDa regulatory subunit (CAPNS1). To date, various expression systems have been developed for producing recombinant calpains for structural and functional studies; however, no low-cost, simple and efficient bacterial expression system for μ-calpain has been available, because the protein forms aggregates. Here, we established an efficient method for producing active recombinant human μ-calpain using an Escherichia coli expression system. This was achieved by co-expressing CAPN1 and CAPNS1 lacking the N-terminal Gly-rich domain (CAPNS1ΔGR) in the SoluBL21 strain. From 1 L of E. coli culture, over 2 and 6 mg, respectively, of μ-calpain and its active-site mutant μ-calpain:C115S (CAPN1:C115S+CAPNS1ΔGR) were purified by two successive column chromatographies. Compared to the native enzyme, the purified μ-calpain showed almost identical properties, demonstrating its suitability for use in structural and functional studies. This is the first report of the bacterial expression and the simple and efficient purification of active recombinant μ-calpain.
authors.affiliation: Calpain Project, 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
authors.forename: Shoji
authors.initials: S
authors.lastname: Hata
authors.forename: Fujiko
authors.initials: F
authors.lastname: Kitamura
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sorimachi
mesh_terms: D001709:Biotechnology / Q000379:methods; D002154:Calpain / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D002154:Calpain; C484806:CAPN1 protein, human