Rapid expression screening of eukaryotic membrane proteins in <i>Pichia pastoris</i>
Bibliographic record
Abstract
The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate-screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N-methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing "jackpot" clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in-culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.
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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 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.002 |
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".