Enhanced expression of engineered ACA-less β-1, 6-N-acetylglucosaminidase (dispersin B) in Escherichia coli
Bibliographic record
Abstract
beta-1,6-N-Acetylglucosaminidase (dispersin B), which cleaves poly-ss-(1,6)-linked N-acetylglucosamine, is encoded by dspB of Aggregatibacter actinomycetemcomitans. To enhance the production of dispersin B, we engineered dspB to transcribe mRNAs devoid of the trinucleotide ACA. Transcription and translation levels of ACA-less and wild-type dspB expressed in Escherichia coli (E. coli) under T5 and T7 promoters were analyzed by real-time RT-PCR and protein quantification, respectively. The ACA-less dspB mRNA level was significantly higher (P < 0.01) and produced 77.6 and 34.9% more dispersin B than wild-type dspB expressed under T7 and T5 promoters, respectively. Dispersin B expression under T7 promoter caused a 98-99.5% drop in the glyceraldehyde-3-phosphate dehydrogenase (gapA) mRNA level, which was not observed with T5 promoter. Fusion of green fluorescent protein (GFP) with dispersin B allowed rapid quantification of dispersin B production by measuring fluorescence intensity in culture broth. Although the cultures containing 0.1% glucose showed sustained increase in dispersin B-GFP production until 12 h, no significant increase in dispersin B activity was observed beyond 4 and 6 h after induction when expressed under T7 and T5 promoters, respectively. This study demonstrates the effectiveness of ACA-less mRNA and the advantage of GFP tagging for enhanced dispersin B production and quantification, which could be adapted for improving the production of other commercially important proteins in E. coli.
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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 | 0.000 | 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 | 0.001 |
| 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 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".