Soluble expression, purification and characterization of the full length IS2 Transposase

A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily.Intractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level.IS2, a 1.3 kb transposable element, is a member of the IS3 family, the largest and most widespread family of insertion sequences (IS) ([1,2]; see also ISfinder: http://www-is.biotoul.fr/is.html webcite). These insertion sequences are characterized by terminal imperfect inverted repeats, the right (IRR) and left (IRL) ends, that flank an internal protein coding sequence (Figure 1a). The latter is comprised of two -1 frameshifted overlapping open reading frames, OrfA and OrfB (Figure 1a, i) and is regulated in IS2 by a weak extended-10 promoter (E-10) promoter (Figure 1b,