Excisionase Purification

Expression and purification of His-tagged Xis and its derivatives. E. coli
strains DH5␣, harboring derivatives of the pYL-XisHR plasmid, and
BL21(DE3), with the pYL-XisHP plasmid, were used for expression of the Xis
mutants and the wild-type Xis protein, respectively. Cells were grown to mid-log
phase in Luria-Bertani medium containing the appropriate antibiotics. Produc-
tion of His-tagged proteins was induced by adding IPTG to a final concentration
of 1 mM, followed by growth for 3 h at 37°C. After centrifugation, harvested cells
were disrupted by treatment in a French pressure cell in 50 mM sodium phos-
phate buffer, pH 7.4, with 300 mM NaCl. Prior to disruption, 250 ␮l of protease
inhibitor cocktail for bacterial cell extracts (Sigma product no. P8465) was added
per gram of cell mass. Cell lysates were centrifuged at 12,000 ϫ g for 30 min, and
the supernatants were mixed with nickel-nitrilotriacetic acid column material
(Qiagen) saturated in 50 mM sodium phosphate buffer, pH 7.4, with 300 mM
NaCl and 10% glycerol. The column was washed extensively, and the His-tagged
proteins were eluted by adding 250 mM imidazole in the same buffer. The total
protein concentrations were measured using the dye-binding assay (3). The
amount of Xis in each protein sample was determined by measuring peaks after
scanning the Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel.