Mutants generated by the insertion of random oligonucleotides into the active site of the .beta.-lactamase gene

Abstract

We have remodeled the gene coding for .beta.-lactamase be replacing DNA at the active site with random nucleotide sequences. The oligonucleotide replacement (Phe66XXXSer70XXLys73) preserves the codon for the active serine-70 but also contains 15 base pairs of chemically synthesized random sequences that code for 2.5 .times. 106 amino acid substituitions. From a population of Escherichia coli infected iwth plasmids containing these random inserts, we have selected seven new active-site mutants that render E. coli resistant to carbenicillin and a series of related analogues. Each of the new mutants contains multiple nucleotide substitutions that code for different amino acids surrounding serine-70. Each of the mutants exhibits a temperature-senstive .beta.-lactamase activity. This technique offers the possibiliity of construcitng alternative active sites in enzymes on the basis of biological selection for functional variants.