PREPARATION OF AN AUTOLYSIS-RESISTANT INTERLEUKIN-1-BETA CONVERTING-ENZYME MUTANT

We describe the expression, purification, and characterization of huma n interleukin-1 beta converting enzyme (ICE) containing an affinity ta g and modified to resist autoproteolysis. The point mutation Asp381 to Glu was added to eliminate the major site of autolytic degradation wh ile maintaining catalytic activity, and an N-terminal polyhistidine ta g was added in place of the ICE pro-region to facilitate purification. N-His (D381E) ICE was expressed in Escherichia coli and purified by n ickel-chelating Sepharose and size-exclusion chromatography (SEC). The enzyme was stabilized greater than 80-fold against autolytic degradat ion relative to wild-type N-His ICE. SDS-PAGE analysis with silver-sta ining revealed no impurities, and 85% of the protein was catalytically active as determined by titration with a novel titrant, PD 163594 lam ino]-4-oxo-5-(2-oxo-2H-chromen-7-yloxypentanoic acid). An oxidized add uct of ICE with glutathione, formed by disulfide rearrangement with ox idized glutathione to inhibit and stabilize the enzyme during purifica tion, was rapidly reduced upon exposure to 5 mM DTT. One mole of gluta thione was released per mole of active enzyme. Of the nine cysteines i n ICE, eight were present in their reduced form in the glutathione add uct, N-His (D381E) ICE cleaved Ac-YVAD-Amc with the Michaelis-Menten p arameters K-M = 14 mu M and k(cat) = 0.7 s(-1), values essentially ide ntical to those reported for enzyme from natural sources.