کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1175584 | 961808 | 2006 | 8 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Variability in the immunodetection of His-tagged recombinant proteins Variability in the immunodetection of His-tagged recombinant proteins](/preview/png/1175584.png)
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 (Epowt) and Epo containing an R103A mutation (EpoR103A). 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 Ni2+– 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.
Journal: Analytical Biochemistry - Volume 359, Issue 2, 15 December 2006, Pages 216–223