Journal
ANALYTICAL BIOCHEMISTRY
Volume 323, Issue 2, Pages 171-179Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2003.08.003
Keywords
epitope tag; immunoaffinity chromatography; green fluorescent protein (GFP); polyol-responsive; Softagl
Funding
- NCI NIH HHS [CA60896, CA14520] Funding Source: Medline
- NIGMS NIH HHS [GM28575] Funding Source: Medline
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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 poly-hydroxylated 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. (C) 2003 Elsevier Inc. All rights reserved.
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