Four derivatives of hen lysozyme, each lacking one native disulfide bond of the four in authentic lysozyme, were produced in Escherichia coli by expressing synthetic mutant genes. In the reoxidation reaction of the reduced derivatives purified from inclusion bodies, the addition of glycerol significantly enhanced the efficiency of folding and 'correct' disulfide bond formation. This enabled simple chromatographical purification of refolded materials. Purified 3SS-derivatives all showed lytic activities and secondary structures comparable to authentic lysozyme, which directly showed that none of the four native disulfide bonds is a prerequisite for 'correct' in vitro folding.