Differential Effect of a His Tag at the N- and C-Termini: Functional Studies with Recombinant Human Serum Transferrin
| Autor: | Valerie C. Smith, Peter J. Halbrooks, Qing-Yu He, Dmitry R. Gumerov, Jeff Hewitt, Anne B. Mason, Ross T. A. MacGillivray, Igor A. Kaltashov, Stephen J. Everse |
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| Rok vydání: | 2002 |
| Předmět: |
Iron
Biology Cleavage (embryo) Biochemistry law.invention Tissue culture law Baby hamster kidney cell Humans Histidine Functional studies DNA Primers chemistry.chemical_classification Base Sequence Transferrin Hydrogen-Ion Concentration HEXA Molecular biology Recombinant Proteins In vitro Kinetics chemistry Recombinant DNA HeLa Cells |
| Zdroj: | Biochemistry. 41:9448-9454 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | Attachment of a cleavable hexa His tag is a common strategy for the production of recombinant proteins. Production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa His tag at the carboxyl terminus, has been described [Mason et al. (2001) Prot. Exp. Purif 23, 142-150]. More recently, hTF-NG with an amino-terminal His tag and a factor Xa cleavage site has been expressed (30 mg/L) in baby hamster kidney cells and purified from the tissue culture medium. Although it is frequently assumed that addition of a His tag has little or no effect on function, this is not always confirmed experimentally. In the present study, in vitro quantitative data clearly shows that the presence of the C-terminal His tag has an effect on the release of iron from recombinant hTF at pH 7.4 and 5.6. Measurement of the rate of release from both the N- and C-lobes is reduced 2-4-fold. These findings provide further compelling evidence that the two lobes communicate with each other and highlight the importance of the C-terminal portion of the C-terminal lobe in this interaction. In contrast to these results, we demonstrate that the presence of a His tag at the N-terminus of hTF has no effect on the rate of iron release from either lobe. In a competition experiment, both unlabeled N- and C-terminal His-tagged constructs were equally effective at inhibiting the binding of radio-iodinated diferric glycosylated hTF from a commercial source to receptors on HeLa cells as the unlabeled recombinant diferric hTF-NG control. Thus, the presence of a His tag at either the N- or C-terminus of hTF-NG has no apparent effect on the ability of these hTF species to bind to transferrin receptors. |
| Databáze: | OpenAIRE |
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