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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Dec;86(23):9094–9098. doi: 10.1073/pnas.86.23.9094

An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase.

A R Oliphant 1, K Struhl 1
PMCID: PMC298440  PMID: 2687873

Abstract

Random-sequence or highly degenerate oligonucleotides have been useful for defining functionally important sequences both in proteins and in nucleic acids. In this approach, such oligonucleotides are used to replace a segment of DNA required for a desired function, and functional sequences are identified by an appropriate genetic or biochemical selection. Here, a collection of 500,000 [corrected] altered beta-lactamase proteins was generated by cloning a mixed-base oligonucleotide in place of the sequences coding for a 17-amino acid portion of the enzyme's active site. Approximately 2000 enzymes from this collection were able to confer ampicillin resistance on Escherichia coli. Fifty-eight of these were chosen for further study after characterization with various beta-lactam substrates. beta-Lactamases having altered specificity against different antibiotics, resistance to the suicide inhibitors clavulanic acid and sulbactam, and temperature-dependent activities were obtained. The amino acid residues responsible for these altered properties as well as for basic enzyme activity are defined. This approach should prove to be an effective and general tool for creating proteins with novel properties, especially in situations in which a high-resolution structure of the protein is not known.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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