Comparative expression of potato proteinase inhibitor type II in an oxidative versus reductive cytosolic environment of Escherichia coli
Bibliographic record
Abstract
Potato proteinase inhibitor type II (PI2) is an enzyme primarily expressed in potato tubers that suppresses the activity of serine proteases such as trypsin and chymotrypsin. PI2 is a disulfide-rich protein that requires an oxidative cytosolic redox state to allow for proper folding in Escherichia coli. In this paper, three different strains of E. coli, BL21 (DE3), SHuffle and Origami 2, were selected for their distinct cytosolic environments. BL21 (DE3) has a reducing cytosolic environment that is not conducive for disulfide bond formation, whereas Origami 2 and SHuffle have oxidative cytosolic environments that promote disulfide bond formation. The objective of this research project was to express and purify PI2 in the three strains of E. coli to compare protein folding of the purified product. Previously, PI2 was cloned into a vector containing a fusion of a maltose-binding protein tag to increase the solubility and to facilitate the purification through amylose affinity chromatography. In the present study, this vector was transformed into the three E. coli strains and the expression of PI2 was induced. PI2 was subsequently purified on an amylose column and the folded state of the purified protein was probed through a limited proteolysis assay. Results indicate that PI2 was expressed in BL21 and SHuffle, but not in Origami 2. However, purified MBP-PI2 protein was only obtained from SHuffle. Limited proteolysis of the purified protein showed that the PI2 purified from SHuffle may have been in the folded state. Therefore, this study demonstrates that PI2 can potentially be expressed in a native conformation in SHuffle.
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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".