Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The ~80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the ~30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of ~80 kDa subunit accumulated at steady state was greatly increased by co-expression of the ~30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.
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Research Article|
March 01 1996
Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system
Sheryl L. MEYER;
Sheryl L. MEYER
*
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
*To whom correspondence should be addressed.
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Donna BOZYCZKO-COYNE;
Donna BOZYCZKO-COYNE
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
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Satish K. MALLYA;
Satish K. MALLYA
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
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Chrysanthe M. SPAIS;
Chrysanthe M. SPAIS
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
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Ron BIHOVSKY;
Ron BIHOVSKY
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
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John K. KAWOOYA;
John K. KAWOOYA
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
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Diane M. LANG;
Diane M. LANG
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
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Richard W. SCOTT;
Richard W. SCOTT
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
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Robert SIMAN
Robert SIMAN
1Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, U.S.A.
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Publisher: Portland Press Ltd
Received:
July 03 1995
Revision Received:
October 23 1995
Accepted:
October 30 1995
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 314 (2): 511–519.
Article history
Received:
July 03 1995
Revision Received:
October 23 1995
Accepted:
October 30 1995
Citation
Sheryl L. MEYER, Donna BOZYCZKO-COYNE, Satish K. MALLYA, Chrysanthe M. SPAIS, Ron BIHOVSKY, John K. KAWOOYA, Diane M. LANG, Richard W. SCOTT, Robert SIMAN; Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system. Biochem J 1 March 1996; 314 (2): 511–519. doi: https://doi.org/10.1042/bj3140511
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