An efficient method to express and refold a truncated human procaspase-9: A caspase with activity toward Glu-X bonds

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• 作者：Ramkrishna Sadhukhan ; Joseph W. Leone ; June Lull ; Zhigang Wang ; Rolf F. Kletzien ; Robert L. Heinrikson and Alfredo G. Tomasselli
• 关键词：Apoptosis ; Apoptosome ; Caspase 9 ; Caspase inhibitors ; Mitochondria ; Procaspase-9
• 刊名：Protein Expression and Purification
• 出版年：2006
• 出版时间：April 2006
• 年：2006
• 卷：46
• 期：2
• 页码：299-308
• 全文大小：302 K

文摘

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, Δ(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 Δ(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 Δ(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 Δ(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.