Immunodetection of recombinant proteins based on antibodies directed against a metal binding peptide engineered for purification by immobilized metal affinity chromatography

Abstract

Recently we reported (Evans, D.B., Tarpley W.G. and Sharma, S.K. (1991) Expression and characterization of chimeric rDNA proteins engineered for purification and cleavage. Protein Expr. Purif. 2, 205–213) a genetically engineered metal binding peptide (mbp) for the purification of recombinant proteins by immobilized metal affinity chromatography (IMAC). Therefore, we have been interested in developing mbp-based immunodetection methods for these engineered proteins. To this end, the following linker peptide containing the mbp (His-Asp-His-Asp-His) was designed, synthesized and conjugated to porcine thyroglobulin: Ac-Cys-Gly-Glu-Glu-His-Asp-His-Asp-His-Pro-His-Leu Rabbits immunized with this conjugate developed antibodies that cross-react with peptides containing the mbp sequence. A number of chimeric recombinant proteins, expressed in E. coli, with and without the mbp portion (His-Asp-his-Asp-His) of the fusion peptide (His-Asp-His-Asp-His-Pro-Phe-His-Leu) were analyzed by ELISA and immunoblotting. Results from these studies show that the anti-mbp antibodies detect chimeric proteins containing the mbp, while chimeric proteins lacking this pentapeptide were negative in both immunodetection techniques. The usefulness of this approach has also been demonstrated in following IMAC purification and enzymatic cleavage of the mbp. These immunodetection techniques utilizing anti-mbp antibodies should be applicable to other proteins engineered to contain the mbp for IMAC purification.