Cloning and expression of a cunner-fish trypsin in bacteria and yeast
Why this work is in the frame
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Bibliographic record
Abstract
Many proteins with special properties have been identified in aquatic organisms. Due to their peculiarity, some of these biomolecules could be used advantageously for some industrial and health care applications. However, the preparation of these biochemicals from its original source is highly impractical and generally unfeasible for commercial purposes. Nevertheless, the advances in DNA manipulation open the possibility of producing the recombinant proteins in different organisms to allow a viable production and extraction on a commercial scale. Among such particular proteins, the cunner-fish trypsin (CFT) is an enzyme with a high potential for exploitation by the food processing industry. The CFT is a cold adapted protease that has a proven ability to inactivate undesirable endogenous enzymes during the processing of some foodstuffs. Thus, the CFT gene was characterized and cloned in E. coli and Pichia pastoris for over-expression. A cDNA library from pancreatic mRNA was screened using a salmon trypsin cDNA probe. Positive clones harboring a cDNA insert of the predicted size were sequenced and a full-length cDNA was obtained that contained an ORF encoding the zymogen and a signal peptide. Multiple alignment of the gene sequence showed 85 to 90 per cent identities with other fish trypsins and the deduced amino acid sequence for the mature enzyme exhibited the entire characteristic features observed in trypsins. E. coli expressed the recombinant CFT (rCFT) at levels higher than 20% in the form of insoluble aggregates, while P. pastoris showed expression levels below 1%. Despite the low expression, the yeast rCFT appeared to be correctly folded and could be partially purified by immobilized nickel affinity chromatography. Trypsin-specific activity was confirmed in the purified rCFT after digestion with bovine enterokinase.
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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 it