Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli
Bibliographic record
Abstract
Listeria monocytogenes is an emerging foodborne pathogen that is responsible for about 28% of the food-related deaths in the United States. It causes meningitis, septicaemia and in pregnant women, abortions and stillbirths. It secretes the toxin listeriolysin O (LLO) that allows the bacteria to enter the cytoplasm of host cells, where they can replicate and cause further infection. The rapid and sensitive detection of LLO in food samples is a key to monitoring and prevention of listeriosis. To facilitate the development of an assay for the specific detection of LLO, a source of LLO is essential. We outline a method of producing a large amount of functional LLO by expressing the hlyA gene (encoding LLO) in Escherichia coli and purifying the recombinant LLO using a one-step purification method. Purification of the protein takes only about 4 h. We compared three different expression constructs for the production of the toxin, which tends to interact strongly with a number of column surfaces. The first construct, using an intein fusion system, could not be purified from the column. The second LLO construct contained an N-terminus His tag; it gave a yield of 3.5-8 mg l(-1). The third contained a C-terminus His tag; it gave a yield of 2.5 mg l(-1) LLO. The purified LLO from the latter two constructs retained its activity at 4 degrees C for over a year as determined by bovine red blood cell hemolysis assay. This paper provides a much-needed, high-yield, one-step purification method of recombinant LLO, and is the first to provide evidence of long-term stability of the toxin for further applications.
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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 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 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".