Overexpression, purification, and characterization of human m-calpain and its active site mutant, m-C105S-calpain, using a baculovirus expression system.
Recombinant human m-calpain was produced in a soluble form at a level of 20 mg/liter of Sf-9 cell culture by the coexpression of recombinant human m-calpain large (m80K) and small (30K) subunits using a baculovirus expression system. The expressed m-calpain was purified by sequential column chromatographies on DEAE-Toyopearl, gel-filtration, and Mono Q by the same method used to purify native m-calpain. The recombinant m-calpain had a specific activity of 691 U/mg and a Ka value (Ca2+ requirement for 50% caseinolysis activity) of 0.4 mM, which are essentially identical to those of native rabbit m-calpain. A mutant m-calpain large subunit, m-C105S-80K, where the active-site cysteine-105 is converted to serine by site-directed mutagenesis, was coexpressed with 30K in Sf-9 cells, purified, and characterized. m-C105S-calpain does not degrade casein nor an artificial tetra-peptide substrate, succinyl-Leu-Leu-Val-Tyr-MCA. Further, it shows no autolytic activity with Ca2+. This is the first report of the large-scale production of a fully active m-calpain species in the baculovirus system.
Masumoto
H
H
Laboratory of Molecular Structure and Function, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0032, Japan. aa67117@hongo.ecc.u-tokyo.ac.jp
Yoshizawa
T
T
Sorimachi
H
H
Nishino
T
T
Ishiura
S
S
Suzuki
K
K
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001665:Binding Sites; D002154:Calpain / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D002154:Calpain