Differential Effect of a His Tag at the N- and C-Termini: Functional Studies with Recombinant Human Serum Transferrin
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- 作者：Anne B. Mason ; Qing-Yu He ; Peter J. Halbrooks ; Stephen J. Everse ; Dmitry R. Gumerov ; Igor A. Kaltashov ; Valerie C. Smith ; Jeff Hewitt ; and Ross T. A. MacGillivray
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：July 30, 2002
- 年：2002
- 卷：41
- 期：30
- 页码：9448 - 9454
- 全文大小：57K
- 年卷期：v.41,no.30(July 30, 2002)
- ISSN：1520-4995
文摘
Attachment of a cleavable hexa His tag is a common strategy for the production of recombinantproteins. Production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containinga 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 afactor Xa cleavage site has been expressed (>30 mg/L) in baby hamster kidney cells and purified fromthe tissue culture medium. Although it is frequently assumed that addition of a His tag has little or noeffect on function, this is not always confirmed experimentally. In the present study, in vitro quantitativedata clearly shows that the presence of the C-terminal His tag has an effect on the release of iron fromrecombinant hTF at pH 7.4 and 5.6. Measurement of the rate of release from both the N- and C-lobes isreduced 2-4-fold. These findings provide further compelling evidence that the two lobes communicatewith each other and highlight the importance of the C-terminal portion of the C-terminal lobe in thisinteraction. In contrast to these results, we demonstrate that the presence of a His tag at the N-terminusof hTF has no effect on the rate of iron release from either lobe. In a competition experiment, bothunlabeled N- and C-terminal His-tagged constructs were equally effective at inhibiting the binding ofradio-iodinated diferric glycosylated hTF from a commercial source to receptors on HeLa cells as theunlabeled recombinant diferric hTF-NG control. Thus, the presence of a His tag at either the N- orC-terminus of hTF-NG has no apparent effect on the ability of these hTF species to bind to transferrinreceptors.