RELATIONSHIP BETWEEN THE OPTIMAL TEMPERATURE FOR OXIDATIVE REFOLDING AND THE THERMAL-STABILITY OF REFOLDED STATE OF HEN LYSOZYME 3-DISULFIDE DERIVATIVES

The temperature dependence of the efficiency of oxidative refolding wa s examined for hen lysozyme three-disulfide derivatives produced in Es cherichia coli. Each derivative was designed to lack one of the four d isulfide bridges in authentic lysozyme: Delta 1 (Cys6-->Ser, Cys127--> Ser), Delta 2 (Cys30-->Ser, Cys115-->Ser), Delta 3 (Cys64-->Ser, Cys80 -->Ser), Delta 4 (Cys76-->Ser, Cys94-->Ser), Delta 2Ala (Cys30-->Ala, Cys115-->Ala), and Delta 4Ala (Cys76-->Ala, Cys94-->Ala). The optimal refolding temperature was lowest for Delta 1 (19 degrees C) and highes t for Delta 4Ala (30 degrees C). The chromatographically purified, com pletely refolded three-disulfide species were not stable above the opt imal refolding temperature in the presence of glutathione. The stabili ty of each of them was determined from the far-UV CD thermal denaturat ion measurement at pH 3.9 in the absence of glutathione, where the den aturation was reversible. The transition temperature was lowest for De lta 1 and highest for Delta 4Ala. Precise values of difference in the transition temperature among the three-disulfide derivatives were foun d to correlate with those in the optimal refolding temperature. Next, the effect of glycerol, which has been shown to increase the refolding efficiency [Sawano et al. (1992) FEBS Lett. 303, 11-14], was examined for Delta 1 in detail. The optimal temperature for refolding increase d by 3-4 degrees C with the increase in glycerol concentration by 10%. The amount of increase in the optimal refolding temperature was nearl y equal to the amount of the increase in thermal stability in the pres ence of glycerol of refolded and purified Delta 1. Taken together, the re exists a parallel relationship for the three-disulfide derivatives between the optimal refolding temperature and the thermal stability of the correctly refolded state. The observation provides the basis for the optimization of the refolding temperature of engineered proteins o f low stability.