Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags

We describe here the use of two newly mapped bovine papillomavirus type I ( BPV-I) E2 protein epitopes as rags. We constucted several vector plasmids f or overexpression as Ic ell as for moderate expression of single- or double -tugged proteins in either Escherichia coli or eukaryotic cells. The new ta gs were fused to several proteins, and the activity of the tugged proteins was tested in different assays. The rags were shown nor to interfere, with the function of these proteins in I vivo and in vitro. Interaction of the m onoclonal antibodies 3F12 and 1E2 with their respective epitopes M as speci fic and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentratio ns rip to 2 M. This permits immunoprecipitation and immunopurification of t he tagged proteins in high-salt buffers and reduction of the nonspecific bi nding of the contaminating proteins. We also provide a protocol for DNA bin ding and DNase I footprinting assays using the tagged resin-bound DNA-bindi ng proteins. The BPV-I E2-derived tags can be recommended as useful tools f or detection and purification of proteins.