Development of an epitope tag for the gentle purification of proteins by immunoaffinity chromatography: application to epitope-tagged green fluorescent protein.
Polyol-responsive monoclonal antibodies (mAbs) are useful tools for the gentle purification of proteins and protein complexes. These are high-affinity mAbs that release the antigen in the presence of a nonchaotropic salt and a low-molecular-weight polyhydroxylated compound (polyol). The epitope for the polyol-responsive mAb NT73, which reacts with Escherichia coli RNA polymerase, was located at the C terminus of the beta' subunit. Using recombinant DNA techniques, we have identified the epitope to be within the 13-amino-acid sequence SLAELLNAGLGGS and have developed an epitope tag that can be fused to a protein of interest for use as a purification tag. This epitope tag (designated Softag1) was fused to either the N or the C terminus of the green fluorescent protein. These tagged proteins were expressed in E. coli, and the tagged proteins were purified from the soluble fraction by a single-step immunoaffinity chromatography procedure. This approach extends the powerful technique of gentle-release immunoaffinity chromatography to many expressed proteins.
Thompson
Nancy E
NE
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706, USA. thompson@oncology.wisc.edu
Arthur
Terrance M
TM
Burgess
Richard R
RR
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D004926:Escherichia coli; D049452:Green Fluorescent Proteins; D007158:Immunologic Techniques; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011108:Polymers / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008164:Luminescent Proteins; D011108:Polymers; C024617:polyol; D049452:Green Fluorescent Proteins