title: Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography.
abstract: The bacterial production of recombinant rat calpain II has been improved greatly by the use of two compatible plasmids for the two subunits. The calpain small subunit C-terminal fragment (21 kDa) was expressed from a new A15-based vector created by cloning T7 control elements into pACYC177. This vector is compatible with the ColE1-based pET-24d(+) vector containing the calpain large subunit, and the yield of calpain activity was increased at least 16-fold by co-expression from these two vectors. A high level of activity was also obtained from a bicistronic construct containing both subunit cDNAs under the control of one T7 promoter. The addition of a C-terminal His-tag to the large subunit simplified purification without affecting subunit association or enzyme activity. The active-site cysteine 105 was mutated to alanine, causing complete loss of activity. The yield of purified C105A-calpain II (80 + 21 kDa) dimer following three column chromatography steps was 10 mg/l of cell culture. This provides a purified calpain, stable to autolysis and oxidation, which is likely to facilitate crystallization in both the presence and absence of calcium.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
authors.forename: J S
authors.initials: JS
authors.lastname: Elce
authors.forename: C
authors.initials: C
authors.lastname: Hegadorn
authors.forename: S
authors.initials: S
authors.lastname: Gauthier
authors.forename: J W
authors.initials: JW
authors.lastname: Vince
authors.forename: P L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002154:Calpain / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D005796:Genes; D005822:Genetic Vectors; D006639:Histidine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D016324:Sequence Tagged Sites
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006639:Histidine; D002154:Calpain