His-tagging involves the addition of a chosen number of histidines to either the N- or C-terminus of the recombinant protein. Poly-histidine tags (His-tags), containing between two (His 2) and ten (His 10) histidine residues, are among the most common affinity tags used for protein purification. Examples of commonly used small peptide affinity tags are poly-arginine (Arg), poly-histidine (His), myc, FLAG and Strep (reviewed in ). There is often a preference for selection of a short affinity tag that can be fused to the target gene more easily using the polymerase chain reaction (PCR). Both detection and purification are greatly simplified by engineering the DNA construct so that the encoded protein is fused to a readily detectable affinant peptide partner, a method designated “affinity tagging”. Methods that will facilitate subsequent protein analysis and purification are of major interest during the initial design of the recombinant protein. Recombinant DNA technology enables the production of large quantities of highly purified and well-characterized proteins. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods.
While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni 2+-NTA resin and by μLC/MS/MS amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti His-tag antibody used. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by SDS-PAGE and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. The cDNA constructs were stably transfected into CHO cells and COS-7 cells. Both were engineered to contain a C-terminal six residue His-tag.
We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo), wild type Epo (Epo wt) and Epo containing an R103A mutation (Epo R103A). Poly-histidine tags (His-tags) are among the most commonly used affinity tags. Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection.
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