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Exploring the functional robustness of an enzyme by in vitro evolution.

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  • 1. Unité de Rétrovirologie Moléculaire, Institut Pasteur, Paris, France.
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    • Martinez MA 1
    (1 author)

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The EMBO Journal, 01 Mar 1996, 15(6):1203-1210
https://doi.org/10.1002/j.1460-2075.1996.tb00461.x PMID: 8635452 PMCID: PMC450021

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Abstract 

The evolution of natural proteins is thought to have occurred by successive fixation of individual mutations. In vitro protein evolution seeks to accelerate this process. RNA hypermutagenesis, cDNA synthesis in the presence of biased dNTP concentrations, delivers elevated mutant and mutation frequencies. Here lineages of active enzymes descended from the homotetrameric 78 residue dihydrofolate reductase (DHFR) encoded by the Escherichia coli R67 plasmid were generated by iterative RNA hypermutagenesis, resulting in >20% amino acid replacement. The 22 residue N-terminus could be deleted yielding a minimum functional entity refractory to further changes, designating it as a determinant of R67 robustness. Complete substitution of the segment still allowed fixation of mutations. By the facile introduction of multiple mutations, RNA hypermutagenesis allows the generation of active proteins derived from extant genes through a mode unexplored by natural selection.

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EMBO J. 1996 Mar 15; 15(6): 1203–1210.
PMCID: PMC450021
PMID: 8635452

Exploring the functional robustness of an enzyme by in vitro evolution.

M A Martinez, V Pezo, P Marlière, and S Wain-Hobson
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Abstract

The evolution of natural proteins is thought to have occurred by successive fixation of individual mutations. In vitro protein evolution seeks to accelerate this process. RNA hypermutagenesis, cDNA synthesis in the presence of biased dNTP concentrations, delivers elevated mutant and mutation frequencies. Here lineages of active enzymes descended from the homotetrameric 78 residue dihydrofolate reductase (DHFR) encoded by the Escherichia coli R67 plasmid were generated by iterative RNA hypermutagenesis, resulting in >20% amino acid replacement. The 22 residue N-terminus could be deleted yielding a minimum functional entity refractory to further changes, designating it as a determinant of R67 robustness. Complete substitution of the segment still allowed fixation of mutations. By the facile introduction of multiple mutations, RNA hypermutagenesis allows the generation of active proteins derived from extant genes through a mode unexplored by natural selection.

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Selected References

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