Fig. 2

The VP1 protein has DNA-binding ability with no sequence specificity. The recombinant GST and GST-fused proteins were prepared by E. coli overexpression and purified through GST affinity chromatography. Purified results were analysed by SDS-PAGE with Coomassie blue staining and Western blotting with an anti-GST monoclonal antibody or anti-C-ter-VP1 polyclonal antibody (a). The purified proteins were used for DNA binding ability by an agarose gel shift assay with different DNA sequences of plasmid preparation of pcDNA3.1 (b), of the pGEM-T easy vector (c), and of pCAV containing the whole CAV genome (d). The binding activity of the VP1 protein was determined by comparing the existence of DNA fragments for the protein-DNA complex and DNA patterns from the blank (no-protein used), negative control (GST only) and positive control (GST-VP3). To confirm the observed DNA migration results that were induced by bound recombinant proteins, the protein-DNA experimental samples were mixed with 1% SDS as a protein denaturant (underline lane-labelled 1% SDS). Lane M, DNA ladder marker. Bold triangles indicated the protein-DNA complex formed by tested proteins and plasmids. Asterisks indicated the two conformations of plasmid DNA, including the relaxed form (Form I), and another was the supercoiled form (Form II)