Recombinant <i>Gaussia</i> Luciferase. Overexpression, Purification, and Analytical Application of a Bioluminescent Reporter for DNA Hybridization
Bibliographic record
Abstract
The cDNA for Gaussia luciferase (GLuc), the enzyme responsible for the bioluminescent reaction of the marine copepod Gaussia princeps, has been cloned recently. GLuc (MW = 19 900) catalyzes the oxidative decarboxylation of coelenterazine to produce coelenteramide and light. We report the first quantitative anaytical study of GLuc and examine its potential as a new reporter for DNA hybridization. A plasmid encoding both a biotin acceptor peptide-GLuc fusion protein as well as the enzyme biotin protein ligase (BPL) is engineered by using GLuc cDNA as a starting template. BPL catalyzes the covalent attachment of a single biotin to the fusion protein in vivo. Purification of GLuc is then accomplished by affinity chromatography using immobilized monomeric avidin. Moreover, the in vivo biotinylation enables subsequent complexation of GLuc with streptavidin (SA), thereby avoiding chemical conjugation reactions that are known to inactivate luciferases. Purified GLuc can be detected down to 1 amol with a signal-to-background ratio of 2 and a linear range extending over 5 orders of magnitude. The background luminescence of coelenterazine is the main limiting factor for even higher detectability of GLuc. Furthermore, the GLuc-SA complex is used as a detection reagent in a microtiter well-based DNA hybridization assay. The analytical range extends from 1.6 to 800 pmol/L of target DNA. Biotinylated GLuc produced from 1 L of bacterial culture is sufficient for 150,000 hybridization assays.
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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) | 0.001 | 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 | 0.001 | 0.000 |
| Research integrity | 0.000 | 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".