An efficient method to express and refold a truncated human procaspase-9: a caspase with activity toward Glu-X bonds.
A truncated form of human procaspase-9 missing the first 111 amino acids, and a variety of mutants derived therefrom, have been expressed in Escherichia coli inclusion bodies. Upon refolding to active enzymes, Delta(1-111) procaspase-9 and mutants were recovered at purity greater than 95% and with a final yield of 20-35 mg/L cell culture. Our active procaspase-9 retains its pro-segment, while undergoing major auto processing at Asp315 and a minor (20%) cleavage at Glu306. This unusual cleavage at a Glu-X bond also took place in the D315E mutant, and we describe herein the inhibitor Z-VAE-fmk that shows enhanced inactivation of procaspase-9 over caspases-3. The bond at Asp330, not processed by procaspase-9, is cleaved by caspase-3 and the resulting procaspase-9 variant, missing the 316-330 bridge, is six times as active as the non-mutated Delta(1-111) proenzyme. A deletion mutant lacking residues 316-330 underwent auto activation by cleavage at Asp315-Ala331 bond. Moreover, substitution of Glu306 by an Asp residue in this mutant led to rapid removal of the peptide spanning Ser307 to Asp330, and resulted in an enzyme that was 7.6 times as active as the non-mutated Delta(1-111) procaspase-9. Finally, replacing both Asp315 and Glu306 with Ala generated a procaspase-9 mutant incapable of auto processing. This single chain procaspase-9 was fully as active as the non-mutated Delta(1-111) enzyme processed at Asp315 or Glu306. Our demonstration that unprocessed procaspase-9 mutants are active as proteases with caspase-type specificity suggests that the role of procaspase-9 in cascade activation of executioner caspases might, in some circumstances, be carried out alone and without association of the apoptosome.
Sadhukhan
Ramkrishna
R
Protein Science, Pfizer Inc. Kalamazoo, MI 49007, USA.
Leone
Joseph W
JW
Lull
June
J
Wang
Zhigang
Z
Kletzien
Rolf F
RF
Heinrikson
Robert L
RL
Tomasselli
Alfredo G
AG
D000592:Amino Acid Metabolism, Inborn Errors; D053453:Caspase 9; D020169:Caspases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion
D011994:Recombinant Proteins; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases