Directed evolution of λ integrase activity and specificity by genetic derepression

Protein Engineering, Design & Selection : PEDS
Jia Wei SiauFarid J Ghadessy

Abstract

Advances in genome engineering are attendant on the development of novel enzyme variants with programed substrate specificities and improved activity. We have devised a novel selection method, wherein the activity of a recombinase deletes the gene encoding an inhibitor of an enzyme conferring a selectable phenotype. By using β-lactamase and the β-lactamase inhibitor protein, the selection couples recombinase activity to Escherichia coli survival in the presence of ampicillin. Using this method, we generated λ integrase variants displaying improved in vitro recombination of a non-cognate substrate present in the human genome. One generalist integrase variant displaying enhanced catalytic activity was further used in a facile, single-step transformation method to introduce transgenes up to 8.5 kb into the unique endogenous attB site of common laboratory E.coli strains.

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Citations

Dec 18, 2015·Nucleic Acids Research·Shree Harsha Vijaya ChandraPeter Dröge
May 11, 2016·Chemical Reviews·Gretchen MeinkeFrank Buchholz
Sep 5, 2020·Stem Cell Research & Therapy·Namrata ChaudhariHarshyaa Makhija
May 3, 2018·Nucleic Acids Research·Adam J BogdanoveBarry L Stoddard
Dec 4, 2020·Nucleic Acids Research·Amer EliasMikhail Kolot
Oct 17, 2020·Scientific Reports·Eugenia VoziyanovaYuri Voziyanov

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