CATALYSIS OF PROTEIN-FOLDING BY AGAROSE-IMMOBILIZED PROTEIN DISULFIDE-ISOMERASE

Protein disulfide isomerase (PDI) catalyzes the formation and rearrangement of disulfide bonds during protein folding. PDI coupled to cyanogen bromide-activated agarose retains its catalytic activity, and a column of this material increases both the rate and the yield for folding disulfide-containing proteins. For reduced, denatured ribonuclease, the overall yield of fully active ribonuclease isolated from the PDI column in one pass was 85-98% of the applied protein. Under the same conditions in the absence of PDI, ribonuclease regained only 16% of its native activity. The oxidative folding of reduced denatured lysozyme is complicated by aggregation so that in the absence of PDI optimal yields of only less than or equal to 25% are obtained at lysozyme concentrations of 1.6 mg/ml. When reduced, denatured lysozyme (1.6 mg/ml) is passed over a PDI column in 1-2 nr urea in the presence of a glutathione redox buffer, the specific activity of the recovered lysozyme is identical to that of the native enzyme and the total recovery of the applied protein is 50-65%. (C) 1994 Academic Press, Inc.