LOOSE FOLDING AND DELAYED OXIDATION PROCEDURES SUCCESSFULLY APPLIED FOR REFOLDING OF FULLY REDUCED HEN EGG-WHITE LYSOZYME

Several factors and/or procedures were examined quantitatively to impr ove the refolding yields of hen egg white lysozyme from its fully dena tured and reduced state. Firstly, we found that refolding treatments w ere better conducted at lower lysozyme concentrations. The refolding y ield decreased from 70% to less than 5% by increasing the lysozyme con centration from 1 to 36 muM in the refolding solution, probably due to aggregation. Secondly, in order to reduce the aggregation and improve the efficiency of refolding, we applied the ''loose folding'' procedu re which required the incubation in the presence of about 2 m urea. Th e refolding of the lysozyme, studied at 17.4 muM, increased the yield to 80% yield in the presence of 2 m urea compared with a 30% yield in the absence of urea. Furthermore, we obtained a dramatic refolding yie ld of more than 95% in an experiment conducted at a concentration of 1 .1 muM lysozyme, in the presence of 2 m urea. Finally, we examined the ''delayed oxidation'' procedure which meant that conformational foldi ng preceded formation of disulfide bonds. The application of this proc edure resulted in increases of 5-10% in the refolding yield. These pro cedures are expected to be useful in improving the refolding yield of precipitated proteins, for example, formed during recombinant DNA prot ein syntheses.