Figure 3.

Expression of mIHF in E. coli and purification of active protein. (a) SDS/PAGE showing purification of mIHF from E. coli. The M. smegmatis mIHF gene was inserted into the expression vector pET21a, and protein expression induced by addition of isopropyl β-d-thiogalactoside (IPTG). Lanes contain markers (M), cells before induction (− IPTG), cells after induction (+ IPTG), ammonium sulfate pellet (pellet), pooled Heparin fractions (Post Hep), and fractions 52–60 obtained by salt elution from a BioCAD carboxymethyl column. (b) mIHF purified from E. coli stimulates recombination. Recombination reactions containing an attP supercoiled DNA, linear attB DNA substrate, L5 Int, and carboxymethyl column fractions (as indicated) were incubated and the products were separated by agarose gel electrophoresis. DNA was identified by ethidium bromide staining and photographed. The recombinant product and the substrates (supercoiled attP DNA and linear attB DNA) are shown.