Avtor/Urednik		Debeljak, Nataša; Feldman, L; Davis, KL; Komel, Radovan; Sytkowski, AJ
Naslov		Variability in the immunodetection of His-tagged recombinant proteins
Tip		članek
Vir		Anal Biochem
Vol. in št.		Letnik 359, št. 2
Leto izdaje		2006
Obseg		str. 216-23
Jezik		eng
Abstrakt		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.
Deskriptorji		RECOMBINANT PROTEINS PEPTIDES HISTIDINE ERYTHROPOIETIN, RECOMBINANT TRANSFECTION POLYMERASE CHAIN REACTION BLOTTING, WESTERN