Mutants generated by the insertion of random oligonucleotides into the active site of the beta-lactamase gene.
We have remodeled the gene coding for beta-lactamase by 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 x 10(6) amino acid substitutions. From a population of Escherichia coli infected with 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-sensitive beta-lactamase activity. This technique offers the possibility of constructing alternative active sites in enzymes on the basis of biological selection for functional variants.
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Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Loeb
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010403:Penicillin Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D001618:beta-Lactamases