Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography.
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.
Elce
J S
JS
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Hegadorn
C
C
Gauthier
S
S
Vince
J W
JW
Davies
P L
PL
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
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006639:Histidine; D002154:Calpain