Improved properties of FLP recombinase evolved by cycling mutagenesis.
The site-specific recombinases FLP and Cre are useful for genomic engineering in many living systems. Manipulation of their enzymatic properties offers a means to improve their applicability in different host organisms. We chose to manipulate the thermolability of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for selection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through cycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of screening at the same stringency to enrich for clones with improved fitness. An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.
Buchholz
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F
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Angrand
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PO
Stewart
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AF
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D019426:Integrases / Q000096:biosynthesis* / Q000737:chemistry; D007763:Lac Operon; D051379:Mice; D016296:Mutagenesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D014764:Viral Proteins
D001425:Bacterial Outer Membrane Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases