Recombinant caspase-3 expressed in Pichia pastoris is fully activated and kinetically indistinguishable from the native enzyme.
Intracellular cysteine proteinases (caspases) play key roles in inflammation and apoptosis. Recombinant caspases are typically produced in Escherichia coli expression systems with the attendant problems of solubilization, re-folding and activation of the protease. Here we describe the expression of hexahistidine-tagged caspase-3 (CPP32/Yama/Apopain) in the methylotropic yeast Pichia pastoris, and the purification of soluble enzyme from yeast lysates using cobalt affinity chromatography. The recombinant protease is fully activated, stable, and cleaves the synthetic substrate DEVD-AFC (Km 16.8 microM) but not YVAD-AFC. It mediates the cleavage of the apoptotic death substrate poly(ADP-ribose) polymerase in cell extracts, but does not cleave pro-interleukin-1beta. It is inhibited by the peptide DEVD-CHO (Ki 2.2 nM), far less efficiently by YVAD-CMK (Ki 0.3 microM), and not detectably by CrmA. By these criteria, recombinant caspase-3 is indistinguishable from native caspase-3 purified from apoptotic cell extracts. Activation of recombinant caspase-3 occurs in yeast in the absence of any intrinsic caspase activity, suggesting that caspase-3 can auto-activate. However, the purified enzyme was incapable of cleaving pro-caspase-3 indicating that autoactivation of caspase-3 in vivo is not likely to occur unless very high concentrations are achieved.
Sun
J
J
Department of Medicine, Monash Medical School, Box Hill Hospital, Australia.
Bottomley
S P
SP
Kumar
S
S
Bird
P I
PI
D053148:Caspase 3; D020169:Caspases; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004789:Enzyme Activation / Q000235:genetics; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012995:Solubility; D013329:Structure-Activity Relationship
D004792:Enzyme Precursors; D011994:Recombinant Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases