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

Protein disulfide isomerase (PDI) catalyzes the formation and rearrang ement of disulfide bonds during protein folding. PDI coupled to cyanog en bromide-activated agarose retains its catalytic activity, and a col umn of this material increases both the rate and the yield for folding disulfide-containing proteins. For reduced, denatured ribonuclease, t he overall yield of fully active ribonuclease isolated from the PDI co lumn in one pass was 85-98% of the applied protein. Under the same con ditions in the absence of PDI, ribonuclease regained only 16% of its n ative activity. The oxidative folding of reduced denatured lysozyme is complicated by aggregation so that in the absence of PDI optimal yiel ds of only less than or equal to 25% are obtained at lysozyme concentr ations of 1.6 mg/ml. When reduced, denatured lysozyme (1.6 mg/ml) is p assed over a PDI column in 1-2 nr urea in the presence of a glutathion e redox buffer, the specific activity of the recovered lysozyme is ide ntical to that of the native enzyme and the total recovery of the appl ied protein is 50-65%. (C) 1994 Academic Press, Inc.