Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system.
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 approximately 80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the approximately 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 approximately 80 kDa subunit accumulated at steady state was greatly increased by co-expression of the approximately 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.
Meyer
S L
SL
Cephalon, Inc., West Chester, PA 19380, U.S.A.
Bozyczko-Coyne
D
D
Mallya
S K
SK
Spais
C M
CM
Bihovsky
R
R
Kaywooya
J K
JK
Lang
D M
DM
Scott
R W
RW
Siman
R
R
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D018076:DNA, Complementary; D011994:Recombinant Proteins; D002154:Calpain; D002118:Calcium