IN SEARCH OF CIRCULAR PERMUTED VARIANTS OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE

A circularized form of a Cys-free mutant of Escherichia coli dihydrofo late reductase (DHFR) was used to search for a proteolytic site that g ave new N- and C-termini on circularized DHFR with enzyme activity. Of the six site-specific proteolytic enzymes tested, three proteases, Ac hromobacter protease I (lysine-specific endopeptidase), asparaginylend opeptidase, and Staphylococcus aureus V8 protease, cleaved a single si te of the circularized DHFR to form circular permuted variants. Twenty -four possible sites for cleavage were found formation of eight circul ar permuted variants was suggested by results of N-terminal sequence a nalysis of the linearized proteins isolated by gel filtration in the p resence of 5 M guanidine hydrochloride. Mapping of the predicted cleav age sites on the DHFR molecule suggested that they mere not all at a s pecific loop and, therefore, there are many possible circular permuted variants.