Preparation of an autolysis-resistant interleukin-1 beta converting enzyme mutant.
We describe the expression, purification, and characterization of human interleukin-1 beta converting enzyme (ICE) containing an affinity tag and modified to resist autoproteolysis. The point mutation Asp381 to Glu was added to eliminate the major site of autolytic degradation while maintaining catalytic activity, and an N-terminal polyhistidine tag was added in place of the ICE pro-region to facilitate purification. N-His (D381E) ICE was expressed in Escherichia coli and purified by nickel-chelating Sepharose and size-exclusion chromatography (SEC). The enzyme was stabilized greater than 80-fold against autolytic degradation relative to wild-type N-His ICE. SDS-PAGE analysis with silver-staining revealed no impurities, and 85% of the protein was catalytically active as determined by titration with a novel titrant, PD 163594 (3-[2-(2-benzyloxycarbonylamino-3-methylbutyrylamino)prop ionylamino]-4- oxo-5-(2-oxo-2H-chromen-7-yloxypentanoic acid). An oxidized adduct of ICE with glutathione, formed by disulfide rearrangement with oxidized glutathione to inhibit and stabilize the enzyme during purification, was rapidly reduced upon exposure to 5 mM DTT. One mole of glutathione was released per mole of active enzyme. Of the nine cysteines in ICE, eight were present in their reduced form in the glutathione adduct. N-His (D381E) ICE cleaved Ac-YVAD-Amc with the Michaelis-Menten parameters K(M) = 14 microM and Kcat = 0.7 s-1, values essentially identical to those reported for enzyme from natural sources.
Dang
L C
LC
BASF Bioresearch Corporation, Worcester, Massachusetts 01605, USA.
Talanian
R V
RV
Banach
D
D
Hackett
M C
MC
Gilmore
J L
JL
Hays
S J
SJ
Mankovich
J A
JA
Brady
K D
KD
D001665:Binding Sites; D020170:Caspase 1; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D009842:Oligopeptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017186:Titrimetry
D003374:Coumarins; D009842:Oligopeptides; D011994:Recombinant Proteins; C096022:acetyl-tyrosyl-valyl-alanyl-aspartyl-7-amino-4-methylcoumarinamide; D003546:Cysteine Endopeptidases; D020170:Caspase 1; D005978:Glutathione