Differential Effect of a His Tag at the N- and C-Termini: Functional Studies with Recombinant Human Serum Transferrin
Bibliographic record
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.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".