TN10 IS10 TRANSPOSASE PURIFICATION, ACTIVATION, AND IN-VITRO REACTION/

We describe a method for the purification of Tn10/IS10 transposase tha t relies on the aggregation of the protein after overexpression in Esc herichia coli. Aggregated transposase was solubilized before the final purification step, a gel-filtration column, using a combination of sa lt and detergent. This procedure is the first reported for the prepara tion of concentrated and active transposase from any IS element. The y ield 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 descr ibe a treatment that activates transposase in either a crude or purifi ed state. This involves dilution into a solution of salt plus organic solvent. In transposition reactions using supercoiled substrate plasmi d, 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 ad ded per transposition event. Maximum conversion of substrate to produc t (40%) was with 18 transposase molecules/transposition event. At high er levels of transposase with a constant amount of substrate, activity was reduced but could be restored by addition of nonspecific DNA. Bot h the specific activity of transposase and the type of products genera ted can be altered by changing in vitro assay conditions. The effects of salts, solvents, and pH value on the reaction are described.