Figure 6From: Soluble expression, purification and characterization of the full length IS2 TransposaseElectrophoretic mobility shift assays. Binding efficiencies of the IS2 OrfAB Transposase derivatives from 22 randomly induced mutants. Reactions were carried out for 30 minutes at 20°C, with 10 nM of 32P-labeled annealed 50-mer oligonucleotides (except where stated in part (f) below) containing the inverted right repeat sequence and 0.11 μM of the partially purified mutant or wild type IS2 OrfAB-GFP protein derivatives (see Methods). Domain locations of the substitutions are color-coded and identified by a single letter code, that is, the binding domain (B) yellow, the leucine zipper-like (L) blue, the catalytic active site (C) green, and the middle interval (M) orange. Reactions were separated on 5% native polyacrylamide gels at 4°C at 120 mA as follows: (a) 450 Vhrs. (b) 420 Vhrs (c) 300 Vhrs (d) 450 Vhrs (e) 450 Vhrs (f) 12% native PAGE for 300 Vhrs using 87-mer annealed oligonucleotides. Binding efficiencies are identified as follows: 5 = Identical to that of the wild type, that is, absence of any dissociation of the complex. 4.5 = a slight loss of compactness of the undissociated complex seen in the wild type control. 4.0 = as in 4.5 but with a faster migrating tail of dissociated complexes. 3.5 = as in 4.0 but with a more prominent faster migrating tail of dissociated complexes. 3.0 = significant loss of compactness of the complex with a small amount of uncomplexed DNA. 2.5 = as in 3.0 but with significantly more uncomplexed DNA. 2.0 = as in 3.0 but mostly composed of uncomplexed DNA. 0.5 = mostly composed of uncomplexed DNA with a small tail of dissociated complex. 0 = no complex formation, identical to that of the protein-free controls (lane 1 in each panel) or the GFP control (part a lane 10). Double mutations are indicated within rectangular boxes. For GMF 18 the operative mutation, L97H, is shown in red (gel c, lane 4). GFP: green fluorescent protein; orf: open reading frame; Vhr: volt hourBack to article page