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Science 2 November 1984:
Vol. 226. no. 4674, pp. 555 - 557
DOI: 10.1126/science.6387910
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Articles

Science, Vol 226, Issue 4674, 555-557
Copyright © 1984 by American Association for the Advancement of Science

articles

Disulfide bond engineered into T4 lysozyme: stabilization of the protein toward thermal inactivation

LJ Perry and R Wetzel

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 wild-type protein. The derivative, T4 lysozyme (Ile3----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 Cys3 and Cys97, one of the two unpaired cysteines of the native molecule. Oxidized T4 lysozyme (Ile3----Cys) exhibited specific activity identical to that of the wild-type enzyme when measured at 20 degrees 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 degrees C.


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