The human T-cell leukemia virus type-2 (HTLV-2) integrase (IN) catalyzes the insertion of the viral genome into the host chromosome. HTLV-2 IN was expressed as an N-terminal hexa-histidine tagged protein in the methylotrophic yeast Pichia pastoris and as a C-terminal hexa-histidine fusion in Escherichia coli. Maximal IN expression was observed at 48 h post-induction for the yeast system and 2 It post-induction for E coli. Effective purification strategies were developed using non-ionic and zwitterionic detergents for initial protein extraction, followed by a one-step nickel-chelating chromatography purification. IN from both sources was routinely greater than 90% pure with yields exceeding 1.5 mg of purified IN per liter of culture for P. pastoris. The relative pI was defined for both INs, pH 5.0-5.4, by 2D-gel electrophoresis. Specific activities for IN purified from E. coli and P. pastoris were calculated from in vitro 3' processing assays and were comparable. In vitro IN assays were also performed to optimize reaction buffer pH and metal concentrations for both 3' processing and strand transfer assays. Strand transfer was optimal from pH 6.2-6.8, more than 1.5 pH units below the optimal 3' processing pH of 8.3. IN from both sources showed no enhancement in activity with MnCl2 concentrations greater than 5 mM. The specific activity of P. pastoris purified IN was 0.35 product (pmol)/h/pg IN, and E coli produced IN was 0.48 product (pmol)/h/mug IN. (C) 2002 Elsevier Science (USA). All rights reserved.