Tn10/IS10 transposase purification, activation, and in vitro reaction.
We describe a method for the purification of Tn10/IS10 transposase that relies on the aggregation of the protein after overexpression in Escherichia coli. Aggregated transposase was solubilized before the final purification step, a gel-filtration column, using a combination of salt and detergent. This procedure is the first reported for the preparation of concentrated and active transposase from any IS element. The yield is 11 mg of purified protein at a concentration of 1 mg/ml from 2.5 g of cells. The procedure can be scaled up with ease. We also describe a treatment that activates transposase in either a crude or purified state. This involves dilution into a solution of salt plus organic solvent. In transposition reactions using supercoiled substrate plasmid, the activity was directly proportional to the amount of transposase added over a wide range of transposase/DNA ratios (0.2-2.0 molecules/DNA substrate molecule). In this range 8 transposase molecules were added per transposition event. Maximum conversion of substrate to product (40%) was with 18 transposase molecules/transposition event. At higher levels of transposase with a constant amount of substrate, activity was reduced but could be restored by addition of nonspecific DNA. Both the specific activity of transposase and the type of products generated can be altered by changing in vitro assay conditions. The effects of salts, solvents, and pH value on the reaction are described.
Chalmers
R M
RM
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138-2092.
Kleckner
N
N
D015139:Blotting, Southern; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D010957:Plasmids; D019895:Transposases
D004251:DNA Transposable Elements; C095509:IS10 transposase; D009713:Nucleotidyltransferases; D019895:Transposases