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Selection and analysis of non-interactive mutants in theEscherichia coli tryptophan synthase a subunit

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Summary

The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in thetrp A gene. Screening of the resulting clones allowed selection of non-interactive mutant α subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: α126 valine (GTG)→glutamic acid (GAG) and α128 valine (GTT)→aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as a66 asparagine (AAC)→aspartic acid (GAC); α109lysine (AAA) →arginine (AGA); α118 cysteine (TGC)→arginine (CGC). Where possible, we individually assessed the importance of these residues in αβ interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.

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Authors and Affiliations

  1. Department of Applied Biochemistry and Food Science, University of Nottingham Faculty of Agriculture and Food Sciences, Sutton Bonington, LE12 5RD, Leicestershire, UK

    Simon Swift & Gordon S. A. B. Steward

  2. Faculty of Biology, Technion-Institute of Technology Haifa, 32000, Israel

    Jonathan Kuhn

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  1. Simon Swift
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  2. Jonathan Kuhn
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  3. Gordon S. A. B. Steward
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Swift, S., Kuhn, J. & Steward, G.S.A.B. Selection and analysis of non-interactive mutants in theEscherichia coli tryptophan synthase a subunit. Molec. Gen. Genet. 233, 129–135 (1992). https://doi.org/10.1007/BF00587570

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  • DOI: https://doi.org/10.1007/BF00587570

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