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Selection strategy for site-directed mutagenesis based on altered beta-lactamase specificity

Academic Article
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Overview

authors

  • Andrews, C. A.
  • Lesley, Scott

publication date

  • June 1998

journal

  • Biotechniques  Journal

abstract

  • Conventional approaches to oligonucleotide-directed mutagenesis rely upon the application of a selection strategy to maximize mutagenesis efficiencies. We have developed a mutagenesis procedure that incorporates a novel antibiotic resistance for selection. The selection involves altering the substrate specificity of TEM-1 beta-lactamase, the enzyme responsible for bacterial resistance to beta-lactam antibiotics such as ampicillin. The gene encoding beta-lactamase is commonly found on cloning and shuttle vectors used in molecular biology. Amino acid substitutions in several active site residues of beta-lactamase result in increased hydrolytic activity against extended-spectrum penicillins and cephalosporins. This increased activity confers a novel resistance specific to the mutant and thus provides the basis of the selection strategy. We describe a simple and efficient mutagenesis procedure and its application to creating a range of oligonucleotide-directed mutants.

subject areas

  • Genetic Vectors
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Substrate Specificity
  • beta-Lactam Resistance
  • beta-Lactamases
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Identity

International Standard Serial Number (ISSN)

  • 0736-6205

PubMed ID

  • 9631188
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Additional Document Info

start page

  • 972

end page

  • 974, 976, 978 passim

volume

  • 24

issue

  • 6

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