Transient gene expression in HEK293 cells: Peptone addition posttransfection improves recombinant protein synthesis
Why this work is in the frame
A frame that forgets how it found something cannot be audited. These are the routes that admitted this work.
Bibliographic record
Abstract
Gene expression by large-scale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins (r-proteins). However, efforts are still needed to optimize production parameters in order to maximize volumetric productivities while maintaining product quality. In this study, transfection efficiency and volumetric productivity following transient gene expression in HEK293 cells were evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) as reporter genes. We show that a single pulse of peptones (protein hydrolysates) to the cultures performed in a low serum (1%, v/v) and in serum-free medium results in a significant increase in volumetric protein productivity. Sixteen peptones from different sources were tested and almost all of them showed a positive effect on r-protein production. This effect, however, is time- and concentration-dependent. By using Tryptone N1 (a casein peptone, TN1) to feed the cultures at 24 h posttransfection (hpt), a 2-fold increase in volumetric SEAP productivity was obtained 5 days posttransfection. This effect was shown to be equal to that obtained when the culture was fed with a supplementary 4% (v/v) of serum. The positive effect of TN1 on protein production was also demonstrated with Tie2 protein ectodomain produced in serum-free medium. HPLC analysis of amino acids consumption/production during control batch and TN1 pulse culture showed some major differences in amino acid metabolism when using TN1 pulse. Asparagine, glycine, histidine, threonine, leucine, and valine show accumulation in the medium over the cultivation period instead of being consumed as observed in unfed sample (except for asparagine, which remained unchanged). Isoleucine, tyrosine, methionine, and phenylalanine all remained unchanged or slightly fluctuated in TN1-fed culture after the feeding pulse, while they were all steadily consumed in the control run. The relative abundance of SEAP's mRNA suggests that the improvement in protein yield results both from an increase of the translational activity and transcription efficiency. Further understanding of mechanisms by which amino acids/peptides regulate transcriptional and translational machinery in mammalian cells should facilitate the design of new strategies for the improvement of r-protein production by large-scale transfection.
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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