Purification and characterization of the active-site-mutated recombinant human mu-calpain expressed in baculovirus-infected insect cells.
Recombinant human mu-calpain whose active site Cys-115 was substituted with Ser was expressed in insect cells using baculovirus system. The mutant mu-calpain, purified using an affinity-column of calpastatin oligopeptides, had no proteolytic activities of autolysis and caseinolysis. The large subunit of the mutant mu-calpain was processed from the 80 kDa form to the 76 kDa form by the wild type calpain, supporting the intermolecular cleavage mechanism of procalpain during activation. Fluorescence polarization analysis revealed that the mutant mu-calpain retained high affinity toward fluorescein-labeled calpastatin domain 1. Fragmentation of the full-length calpastatin by the wild type calpain was enhanced by pre-incubating the inhibitor with the mutant calpain. The recombinant mutant calpain was suggested to retain the integrity of the high ordered structure of the wild type calpain.
Hitomi
K
K
Laboratory of Molecular and Cellular Regulation, Nagoya University, Japan.
Uchiyama
Y
Y
Ohkubo
I
I
Kunimatsu
M
M
Sasaki
M
M
Maki
M
M
D000818:Animals; D001329:Autolysis; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics*; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002846:Chromatography, Affinity; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology
D002135:Calcium-Binding Proteins; D002364:Caseins; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D011994:Recombinant Proteins; C033668:calpastatin; D002154:Calpain; C047018:procalpain; D002118:Calcium