Small scale expression and purification protocol of A71 inactive 2A protease with mutation C110A to preserve VP1-2A junction v1
Bibliographic record
Abstract
This protocol describes the small-scale expression and purification of Enterovirus A71 inactive 2A protease bearing the C110A mutation to preserve the VP1-2A junction. The protein is expressed as an N-terminal His6-SUMO fusion in E. coli BL21(DE3)-RR cells using TB autoinduction media, yielding approximately 6 mg/L of culture. The purification strategy employs immobilized metal affinity chromatography (IMAC) for initial protein capture, followed by tag cleavage using His-SENP1 protease and reverse IMAC to remove the cleaved affinity tag and uncleaved protein. Final polishing is achieved through size exclusion chromatography, producing >95% pure untagged 2A protease. From 6 L of culture, typical yields are approximately 50 mg of pure protein.
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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 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.000 | 0.002 |
| Insufficient payload (model declined to judge) | 0.005 | 0.007 |
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".