[Prokaryotic expression of DNA recombinase FLP and its purification with enzymatic activity].
DNA recombinase FLP gene exists on the 2 micro plasmid of Saccharomyces cerevisiae. Recombinase FLP could recognize an FRT site composed of 34bp and function the sequences for exchange, recombination, deletion and reversion between the two orientated FRT sites. These functions are highly recognized by molecular biologists and biotechnology engineers for theoretic and applicable technology studies. This work constructed a prokaryotic over-expressed vector harboring FLP gene nominated as pQE30-flpe and established its over-expression culture system in which recombinase FLP could be efficiently expressed in E. coli strain M15. Purification procedures for high purity and active FLP are established through combination of ammonium sulfate precipitation with a 0.5-1.0 mL micro-column technique of Ni affinity chromatography with gradient elution. To verify the recombinase activity of purified FLP, substrate vectors, sequence donor vector (pUC18-FRT-gfp-FRT) and sequence accepting vector (pET30a-FRT) are constructed with various number, orientation of FRTs harboring the GFP gene for the expression of visible assay of the functions of recombination, exchange and deletion. Results showed that the system not only over expressed recombinase FLP in prokaryotic E. coli, but also efficiently purified the enzyme with a higher activity of the function of recombination, exchange and deletion. The system and the method are easily implemented and feasibly manipulated for theoretic study and biotechnology application.
Gai
Ying
Y
College of Life Science and Biotechnology, Beiing Forestry University, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of State Forestry Administration, Beijng 100083, China.
Wang
Wen-Qi
WQ
Lu
Hai
H
Jiang
Xiang-Ning
XN
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology*