A Fluorescent Protein-Based Biological Screen of Proteinase Activity
Bibliographic record
Abstract
A cell-based fluorescent protein reporter assay for proteinase activity amenable to high-throughput applications was developed. This assay is based on Förster resonance energy transfer (FRET) between 2 variants of the green fluorescent protein connected by a short cleavable linker and expressed in Escherichia coli as tagged proteins. A library to assay proteinase specificity was generated by randomizing a portion of the linker using PCR. The library could be grown in microplates, allowing cells to be lysed in situ and substrate cleavage to be monitored through loss of FRET signal using a plate reader. Progress curves were generated to estimate cleavage efficiency, facilitating the identification of well-cleaved substrates. The polyhistidine-tagged fluorescent substrates could then be purified and used for further characterization. To establish the general utility of the screen, it was used to demonstrate that the cysteine proteinase of the hepatitis A virus, 3C(pro), prefers Ile, Val, or Leu at the P(4) position of the cleavage sequence and Gly, Ser, or Ala at the P'(1) position. The assay can also be used to screen small-molecule libraries for inhibitors.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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 source (direct Gemma or distilled Codex), 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".