Jia Wei Siau, Sharon Chee, Harshyaa Makhija, Cho Mar Myint Wai, Shree Harsha Vijaya Chandra, Sabrina Peter, Peter Dröge, Farid J. Ghadessy, Directed evolution of λ integrase activity and specificity by genetic derepression, Protein Engineering, Design and Selection, Volume 28, Issue 7, July 2015, Pages 211–220, https://doi.org/10.1093/protein/gzv015
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-cog- nate 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 trans- genes up to 8.5 kb into the unique endogenous attB site of common laboratory E.coli strains.
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