Improved properties of FLP recombinase evolved by cycling mutagenesis.

Abstract:

: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.

journal_name

Nat Biotechnol

journal_title

Nature biotechnology

authors

Buchholz F,Angrand PO,Stewart AF

doi

10.1038/nbt0798-657

subject

Has Abstract

pub_date

1998-07-01 00:00:00

pages

657-62

issue

7

eissn

1087-0156

issn

1546-1696

journal_volume

16

pub_type

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