Differential Effect of a His Tag at the N- and C-Termini:  Functional Studies with Recombinant Human Serum Transferrin

Abstract

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.

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

“…In fact the side chain of Gln 20 in the A molecule lies within hydrogen bonding distance of Cys 177 and Cys 179 in the B molecule. Additionally, the side chain of Glu 13 in molecule A interacts with the side chain of Arg 124 in molecule B. This is of interest, because Arg 124 is the critical anion binding residue in the N-lobe of hTF, whose important role in iron uptake and release has been extensively studied (6, 19, 50 -52).…”

“…Production of hTF-NG-To produce recombinant non-glycosylated hTF (hTF-NG) with SeMet substituted for methionine, baby hamster kidney cells transfected with the pNUT plasmid containing the sequence of the N-His-tagged hTF-NG were placed into four expanded surface roller bottles (13). Addition of culture media containing SeMet results in a significant deterioration of the cells within 24 -48 h. To maximize the incorporation, medium containing SeMet was added when production of hTF was at a maximum as determined by a competitive immunoassay (29).…”

Effects of in Vitro Glycation on Fe3+ Binding and Fe3+ Isoforms of Transferrin

“…This issue has led to abundant but often contradictory literature. The N-and C-terminal binding sites have similar structures, but they are not equivalent in terms of Fe 3ϩ uptake and release because they differ in their accessibility to iron chelates, binding strength, spectroscopic properties, kinetic lability, and response to changes in pH (29,30,37,38 ). For example, it is known that the C-terminal site can hold Fe 3ϩ at a lower pH (39,40 ).…”

Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity

“…Moreover, crystal structure studies showed that these tags generally have no significant effect on the structure of the tagged protein in comparison to the native protein [47]. However, previous studies demonstrated that the His-tag fused to the C-terminus of a recombinant human serum transferrin [48], as well as of a recombinant molybdoenzyme [49] negatively affected their function. In contrast, a BmK insect toxin was positively affected E total is the sum of the interaction energy between the substrate and the receptor (E steric + E HB + E internal ).…”