Site-Directed Mutagenesis Using Altered /gb-Lactamase Specificity

  • Christine A. Andrews
  • Scott A. Lesley
Part of the Methods in Molecular Biology™ book series (MIMB, volume 182)


Site-directed mutagenesis (SDM) is a powerful tool for the study of gene expression/regulation and protein structure and function. Hutchinson et al. (1) developed a general method for the introduction of specific changes in DNA sequence, which involves hybridization of a synthetic oligonucleotide (ON) containing the desired mutation to a single-stranded DNA (ssDNA) target template. Following hybridization, the oligonucleotide is extended with a DNA polymerase to create a double-stranded structure. The heteroduplex DNA is then transformed into an Escherichia coli, in which where both wild type and mutant strands are replicated. In the absence of any selection this method is very inefficient, often resulting in only a few percent of mutants obtained. Various strategies of selection have since been developed, which can increase mutagenesis efficiencies well above the theoretical yield of 50%. The methods of Kunkel (2), Eckstein (3), and Deng (4,5) employ negative selection against the wild-type DNA strand, in which the parental DNA is selectively degraded, either by growth in an alternate host strain, or by digestion with a nuclease or restriction enzyme. The methods of Lewis and Thompson (6) and Bonsack (7) utilize antibiotic resistance to positively select for the mutant DNA strand. This chapter describes a method for the positive selection of mutant strand DNA, which relies on the altered activity of the enzyme β-lactamase against extended spectrum cephalosporins (8).


Isoamyl Alcohol Transformation Reaction JM109 Cell Annealing Reaction Extend Spectrum Cephalosporin 
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  1. 1.
    Hutchinson, C. A., Phillips, S., Edgell, M. H., Gillam, S., Jahnke, P., and Smith, M. (1978) Mutagenesis at a specific position in a DNA sequence J. Biol. Chem. 253, 6551–6560.Google Scholar
  2. 2.
    Kunkle, T. A. (1985) Rapid and efficient site-specific mutagenesis without phe-notypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492.CrossRefGoogle Scholar
  3. 3.
    Taylor, J. W., Ott, J., and Eckstein, F. (1985) The rapid generation of oligonucle-otide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 13, 8764–8785.Google Scholar
  4. 4.
    Deng, W. P. and Nickoloff, J. A. (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal. Biochem. 200, 81–88.PubMedCrossRefGoogle Scholar
  5. 5.
    Nickoloff, J. A., Miller, E. M., Deng, W. P., and Ray, F. A. (1996) Site-directed mutagenesis of double-stranded plasmids, domain substitution, and marker rescue by co-mutagenesis of restriction sites, in Basic DNA and RNA Protocols (Harwood, A., ed.), Humana, Totowa, NJ, pp. 455–468CrossRefGoogle Scholar
  6. 6.
    Lewis, M. K. and Thompson, D. V. (1990) Efficient site-directed in vitro mutagenesis using ampicillin selection. Nucleic Acids Res. 18, 3439–3443.PubMedCrossRefGoogle Scholar
  7. 7.
    Bohnsack, R. N. Site-directed mutagenesis using positive antibiotic selection, in Methods in Molecular Biology, vol. 57: In Vitro Mutagenesis Protocols (Trower, M. K., ed.), Humana, Totowa, NJ, pp. 1–12.Google Scholar
  8. 8.
    Andrews, C. A. and Lesley, S. A. (1998) Selection strategy for site-directed mutagenesis based on altered ?-lactamase specificity. Biotechniques 24, 972–977.PubMedGoogle Scholar
  9. 9.
    Cantu, III, C., Huang, W., and Palzkill, T. (1996) Selection and characterization of amino acid substitutions at residues 237-240 of TEM-1 ?-lactamase with altered substrate specificity for aztreonam and ceftazidime. J. Biol. Chem 271, 22,538–22,545.PubMedCrossRefGoogle Scholar
  10. 10.
    Venkatachalam, K. V., Haung, W., LaRocco, M., and Palzkill, T. (1994) Characterization of TEM-1 ?-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime. J. Biol. Chem. 269, 23,444–23,450.PubMedGoogle Scholar
  11. 11.
    Delaire, M. Labia, R., Samama, J. P., and Masson, J. M. (1991) Site-directed mutagenesis on TEM-1 ?-lactamase: role of Glu 166 in catalysis and substrate binding. Protein Eng. 4, 805–810.PubMedCrossRefGoogle Scholar
  12. 12.
    Imtiaz, U., Manavathu, E., Mobashery, S., and Lerner, S. A. (1994) Reversal of a clavulanate resistance conferred by a Ser-244 mutant of TEM-1 b-lactamase as a result of a second mutation (arg to Ser at position 164) that enhances activity against ceftazidime. Antimicrob. Agents Chemother. 38, 1134–1139.PubMedCrossRefGoogle Scholar
  13. 13.
    Matagne, A., Misselyn-Bauduin, A. Joris, B., Erpicum, T., Grainer, B., and Frere, J. M. (1990) The diversity of the catalytic properties of class A ?-lactamases Biochem. J. 265, 131–146.PubMedGoogle Scholar
  14. 14.
    Palzkill, T. and Botstein, D. (1992) Identification of amino acid substitutions that alter the substrate specificity of TEM-1 ?-lacatmase. J. Bacteriol. 174, 5237–5243.PubMedGoogle Scholar
  15. 15.
    Palzkill, T. and Botstein, D. (1992) Probing ?-lactamase structure and function using random replacement mutagenesis. Proteins Struct. Funct., Genet. 14, 29–44.CrossRefGoogle Scholar
  16. 16.
    Palzkill, T., Le, Q. Q., Venkatachalam, K. V., La Rocco, M. and Ocera, H. (1994) Evolution of antibiotic resistance: several different amino acids in an active site loop alter the substrate profile of ?-lactamase. Mol. Microbiol. 12, 217–229.PubMedCrossRefGoogle Scholar
  17. 17.
    Petit, A., Maveyraud, L., Lenfant, F., Samama, J. P., Labia, R., and Masson, J. M. (1995) Multiple substitutions at position 104 of ?-lactamse TEM-1: assessing the role of this residue in substrate specificity. Biochem. J. 305, 33–40.PubMedGoogle Scholar
  18. 18.
    Ambler, R. P. (1979) Amino acid sequences of ?-lactamases, in ?-Lactamase (Hamilton-Miller, J. M. T. and Smith, J. T., eds.), Academic Press, London.Google Scholar
  19. 19.
    Kramer, B., Kramer, W., and Fritz, J. J. (1984) Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch repair system of E. coli. Cell 38, 879–887.Google Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Christine A. Andrews
    • 1
  • Scott A. Lesley
    • 2
  1. 1.Protein Expression/Enzymology, Research and DevelopmentPromega CorporationMadison
  2. 2.Protein Expression and AnalysisGenomics Institute of the Novartis Research FoundationSan Diego

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