Disulfide Bond Engineered into T4 Lysozyme: Stabilization of the Protein toward Thermal Inactivation

By recombinant DNA techniques, a disulfide bond was introduced at a specific site in T4 lysozyme, a disulfide-free enzyme. This derivative retained full enzymatic activity and was more stable toward thermal inactivation than the wildtype protein. The derivative, T4 lysozyme (Ile$^{3}\rightarrow $ Cys),, was prepared by substituting a Cys codon for an Ile codon at position 3 in the cloned lysozyme gene by means of oligonucleotide-dependent, site-directed mutagenesis. The new gene was expressed in Escherichia coli under control of the (trp-lac) hybrid tac promoter, and the protein was purified. Mild oxidation generated a disulfide bond between the new Cys$^{3}$ and Cys$^{97}$, one of the two unpaired cysteines of the native molecule. Oxidized T4 lysozyme (Ile$^{3}\rightarrow $ Cys) exhibited specific activity identical to that of the wild-type enzyme when measured at 20 ° C in a cell-clearing assay. The cross-linked protein was more stable than the wild type during incubation at elevated temperatures as determined by recovered enzymatic activity at 20 °C.