Differential effect of a his tag at the N- and C-termini: functional studies with recombinant human serum transferrin.
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.
Mason
Anne B
AB
Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405, USA. amason@zoo.uvm.edu
He
Qing-Yu
QY
Halbrooks
Peter J
PJ
Everse
Stephen J
SJ
Gumerov
Dmitry R
DR
Kaltashov
Igor A
IA
Smith
Valerie C
VC
Hewitt
Jeff
J
MacGillivray
Ross T A
RT
D001483:Base Sequence; D017931:DNA Primers; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D014168:Transferrin; D006639:Histidine; D007501:Iron