title: Variability in the immunodetection of His-tagged recombinant proteins.
abstract: Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. 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)). Both were engineered to contain a C-terminal six residue His-tag. The cDNA constructs were stably transfected into Chinese hamster ovary (CHO) cells and COS-7 cells. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni(2+)- nitrilotriacetic acid resin and by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometery amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti-His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods.
authors.affiliation: Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, 330 Brookline Ave., W/BL 548, Boston, MA 02215, USA.
authors.forename: Natasa
authors.initials: N
authors.lastname: Debeljak
authors.forename: Laurie
authors.initials: L
authors.lastname: Feldman
authors.forename: Kerry L
authors.initials: KL
authors.lastname: Davis
authors.forename: Radovan
authors.initials: R
authors.lastname: Komel
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Sytkowski
mesh_terms: D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007158:Immunologic Techniques; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D004921:Erythropoietin; D006639:Histidine