Large scale EV-A71 2A protease mature and precursor expression setup and operation of Single Use Bubble Column Reactors: Litre-Scale Expression of Recombinant Proteins for Structural Biology and Drug Design (SBDD) v1
Bibliographic record
Abstract
This protocol describes a single-use Bubble Column Reactor (suBCR) system for large-scale recombinant protein production in E. coli, addressing limitations of traditional shake-flask methods for structure-based drug discovery. The system enables parallel 1-liter batch cultivation using disposable bioreactor bags in a heated water bath with controlled aeration. Two enterovirus 2A protease variants were successfully expressed: wild-type Human Coxsackievirus A16 2A protease (Addgene 228632) and an inactive Coxsackievirus A71 2A protease with C110A mutation (Addgene 228633).The C110A mutation preserves the VP1-2A junction while eliminating catalytic activity, providing active and inactive variants for comparative studies that are suitable for crystallography. Cultures are grown at 37°C for 4 hours until exponential phase, then expressed overnight at ambient temperature (25-27°C) using auto-induction Terrific Broth media. The system achieved high yields with culture volumes of 5-22.6 L producing wet cell weights of 163.2-225.6 g. Quality control via nickel-magnetic bead purification and SDS-PAGE confirmed successful overexpression of both constructs. This scalable, cost-effective approach significantly advances protein production methodology for structural biology and drug discovery, offering increased throughput and reduced labor compared to conventional methods while providing sufficient protein quantities for crystallographic studies and antiviral drug development.
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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 | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.002 | 0.003 |
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".