Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags.
We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays. The tags were shown not to interfere with the function of these proteins in vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprinting assays using the tagged, resin-bound DNA-binding proteins. The BPV-1 E2-derived tags can be recommended as useful tools for detection and purification of proteins.
Kaldalu
N
N
Tartu University, Estonia.
Lepik
D
D
Kristjuhan
A
A
Ustav
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D004247:DNA / Q000378:metabolism; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000276:immunology*; D000939:Epitopes; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D014764:Viral Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; C051550:E2 protein, Bovine papillomavirus; D000939:Epitopes; D011506:Proteins; D014764:Viral Proteins; D004247:DNA