Purification of the FLP site-specific recombinase by affinity chromatography and re-examination of basic properties of the system.
The FLP protein, a site-specific recombinase encoded by the 2 micron plasmid of yeast, has been purified to near homogeneity from extracts of E. coli cells in which the protein has been expressed. The purification is a three column procedure, the final step employing affinity chromatography. The affinity ligand consists of a DNA polymer with multiple FLP protein binding sites arranged in tandem repeats. This protocol yields 2 mg of FLP protein which is 85% pure. The purified protein is highly active, stable for several months at -70 degrees C and free of detectable nucleases. The molecular weight and N-terminal sequence are identical to that predicted for the FLP protein by the DNA sequence of the gene. Purified FLP protein primarily, but not exclusively, promotes intramolecular recombination. Intermolecular recombination becomes the dominant reaction when E. coli extracts containing no FLP protein are added to the reaction mixture. These extracts are not specifically required for recombination, but demonstrate that some properties previously attributed to FLP protein can be assigned to contaminating proteins present in E. coli.
Meyer-Leon
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L
Gates
C A
CA
Attwood
J M
JM
Wood
E A
EA
Cox
M M
MM
D000345:Affinity Labels; D001483:Base Sequence; D002846:Chromatography, Affinity; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification*; D004926:Escherichia coli / Q000032:analysis; D005656:Fungal Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000345:Affinity Labels; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase