Detection of histidine-tagged fusion proteins by using a high-specific mouse monoclonal anti-histidine tag antibody

摘要

Recombinant proteins are utilized for various purposes in molecular biology such as the production of antibodies or investigation of the mechanism of protein-nucleic acid or protein-protein interactions. Many prokaryotic expression vectors have been established enabling synthesis of the protein of interest as a fusion with a peptide, thus facilitating purification. Expression of recombinant proteins in Escherichia coli as a fusion protein with neighbouring histidine residues is one of the most popular methods (1; Diagen), because these proteins have useful attributes. The affinity of the histidine tag motif to Ni~(2+) ions by chelation is strong and selective enough to enable purification of the protein to homogeneity by affinity chromatography on aNi~(2+)-NTA adsorbant (1). Additionally, an enterokinase cleavage site engineered between the N- or C-terminus of the protein of interest and the polyhistidine fusion partner facilitates the removal of the tag. A drawback of the polyhistidine fusion system so far may be the lack of sera specific for the affinity tag. To circumvent this limitation we raised mouse monoclonal anti-histidine tag antibodies by immunizing mice with mixtures of histidine-tagged proteins, engineered in our laboratory using standard immunizing protocols (2). Such an antibody allows the detection of proteins with a histidine tag without the need to rase specific sera against the protein of interest. Here we present data obtained using the anti-histidine tag antibody mAb 13/45/31 (subclass IgG2a).