Cited by 200Evolution and Genetic DynamicsAntibiotic Resistance in BacteriaEnzyme Catalysis and Immobilization

The effect of high-frequency random mutagenesis on in vitro protein evolution: a study on TEM-1 β-lactamase 1 1Edited by A. R. Fersht

Zaccolo, Manuela, Gherardi, Ermanno·1999·Journal of Molecular Biology

Abstract

For a number of years a major limitation in genetic analysis of protein function has been the inability to introduce multiple substitutions at distant sites that would enable the selection of clusters of mutations required for improved or novel biological functions. In order to achieve this, we have recently developed a novel mutagenesis procedure in which the triphosphate derivatives of a pyrimidine (6-(2-deoxy-beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one; dP) and a purine (8-oxo-2'-deoxyguanosine; 8-oxodG) nucleoside analogue are employed in DNA synthesis reactions in vitro. The procedure allows control of the mutational load and can yield frequencies of amino acid residue substitutions at least one order of magnitude greater than those previously achieved. Here we report the results of an experiment in which we have hypermutated the bacterial enzyme TEM-1 beta-lactamase and selected small pools (<1.5x10(5)) of clones for enzymatic activity against the beta-lactam antibiotic cefotaxime. The experiment resulted in the isolation of a number of TEM-1 mutants with greatly improved activity against cefotaxime. Among these, clone 3D.5 (E104K:M182T:G238S) exhibited a minimum inhibitory concentration for cefotaxime 20,000-fold higher than wild-type TEM-1 and a catalytic efficiency (kcat/Km) 2383 times higher than the wild-type enzyme. Thus, small pools of hypermutated sequences enabled the selection of one of the most active extended beta-lactamases described so far. These results argue against the accepted view that multiple rounds of low-rate mutagenesis and stepwise selection are essential for in vitro protein evolution and extend the scope of directed molecular evolution to proteins for which no genetic selection is available.

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Crossref · 10.1006/jmbi.1998.2262

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  • The mutagenetic PCR method introduces an approximate 5% error rate to the final PCR product.

    “their methods employ mutagenetic PCR on template strands which introduces an approximate 5% error rate to the final PCR product12 while maintaining a conserved PCR primer region.”

    From Probing the physical limits of reliable DNA data retrieval. (2020) · cites this paper as [12]

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Authors
Zaccolo, M., & Gherardi, E.
Affiliations
Medical Research Council (GB)
Corresponding author
Gherardi, Ermanno
Venue
Journal of Molecular Biology
Publisher
Elsevier BV
Published
1999-01
Volume
285
Issue
2
Pages
775-783
ISSN
0022-2836 (ISSN-L)
OpenAlex
W2018627617
Cited by
200
References
44
Citations by year
2026: 3 · 2025: 3 · 2024: 1 · 2023: 4 · 2022: 4 · 2021: 4 · 2020: 5 · 2019: 4 · 2018: 3 · 2017: 2 · 2016: 6 · 2015: 5 · 2014: 7 · 2013: 10 · 2012: 8
Type
book review
Language
en
Open access
closed
Locations
  • Journal of Molecular Biology (publishedVersion)
  • PubMed (publishedVersion)
Funders
Royal Society
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Medicine › Antibiotic Resistance in Bacteria
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
in vitro protein evolution, TEM-1 beta-lactamase, cefotaxime resistance, hypermutation, 8-hydroxy-2'-deoxyguanosine, catalytic efficiency, beta-lactamase
MeSH terms
beta-Lactamases, Cefotaxime, Mutagenesis, Directed Molecular Evolution

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