CIRCULARLY PERMUTED DIHYDROFOLATE-REDUCTASE OF ESCHERICHIA-COLI HAS FUNCTIONAL-ACTIVITY AND A DESTABILIZED TERTIARY STRUCTURE

Three circularly permuted variants of Escherichia coli dihydrofolate r eductase genes were constructed. Linkers coding tri- and pentapeptides were used to connect the natural 5'- and 3'-terminal ends, Only one v ariant of circularly permuted protein with tripeptide linker and the c leavage of the peptide bond between 107 and 108 amino acid residues wa s produced in a good yield. The expressed protein was insoluble in the cells, but at pH 8.0 and higher the isolated protein was soluble. Enz ymatic assay and physical studies have shown that permuted dihydrofola te reductase has a destabilized tertiary structure. Only the addition of the natural substrates or inhibitors lead to the protein with the n ative-like structure and functional activity.