Intein-mediated rapid purification of Cre recombinase.
Cre recombinase produced by bacteriophage P1 catalyzes site-specific recombination of DNA between loxP recognition sites in both prokaryotic and eukaryotic cells and has been widely used for genome engineering and in vitro cloning. Recombinant Cre has been overproduced in Escherichia coli and its purification involves multiple steps. In this report, we used an "intein" fusion system to express Cre as a C-terminal fusion to a modified protein splicing element, i.e., intein. The modified intein contained a Bacillus circulans chitin-binding domain which allowed binding of the fusion protein on a chitin column and could be induced to undergo in vitro peptide bond cleavage which specifically released Cre from the column. Using the intein system, we have obtained highly pure nontagged Cre after just a single chromatographic step, which corresponded to approximately 80% recovery and 27-fold purification. The activity of the purified Cre was determined in an in vitro assay system and was found to remain stable over a period of more than 6 months.
Cantor
E J
EJ
New England Biolabs, Inc., 32 Tozer Road, Beverly, Massachusetts 01915, USA.
Chong
S
S
D001407:Bacillus / Q000737:chemistry / Q000235:genetics; D017101:Bacteriophage P1 / Q000201:enzymology* / Q000235:genetics; D002686:Chitin / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D002686:Chitin; C045073:Cre recombinase; D019426:Integrases