HEK 293 cells | EPFL Graph Search

Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific immortalised cell line derived from a spontaneously miscarried or aborted fetus or human embryonic kidney cells grown in tissue culture taken from a female fetus in 1973. HEK 293 cells have been widely used in cell biology research for many years, because of their reliable growth and propensity for transfection. They are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy as well as safety testing for a vast array of chemicals. 293T (or HEK 293T) is a derivative human cell line that expresses a mutant version of the SV40 large T antigen. It is very commonly used in biological research for making proteins and producing recombinant retroviruses. History HEK 293 cells were generated in 1973 by transfection of cultures of normal human embryonic kidney cells with sheared adeno

Two strategies to improve transient gene expression in HEK293E and/or CHO-DG44 cells

Guillaume Lüthi

Transient gene expression (TGE) is based on episomal DNA expression. This technique is used for the production of recombinant proteins that can be used as therapeutics. TGE is for example extremely useful for shortening the time between candidate proteins screening and their production. Understanding and optimizing TGE is a major concern in the field of recombinant proteins. Thus, we selected here two different approaches to further improve TGE. First, we studied effect of signal peptide (SP) modification on TNFR-Fc protein in transiently transfected HEK293E and CHO-DG44 cells. Secondly, we studied effect of Valproic acid on the production of TNFR-Fc protein in HEK293E cells and on its influence on plasmid methylation. Modification of TNFR-Fc was done with eighteen others SPs. Artificial SPs exhibiting different scores using a prediction method and SPs coming from naturally secreted proteins were tested. None of them could induce a better secretion than the native SP. Nevertheless, they all showed comparable secretion abilities, except one that could not induce protein secretion. We showed here that SP modification could not increase protein secretion in HEK293E or in CHO-DG44. But also that without a valid SP, protein secretion is inhibited. Addition of VPA in HEK293E cells transfected with pXLG-A3 plasmid increased IgG productivity by 11 fold. Nevertheless, we showed here that VPA did not increase TNFR-Fc productivity to the same extent (1.2 fold) even by optimization of its concentration. Furthermore, we investigated the effect of plasmid methylation (TNFRFc and IgG) on TGE using VPA. We found that IgG expression was less influenced by this modification than TNFR-Fc. In fact, TNFR-Fc plasmid methylation drastically decreased specific productivity (0.2 fold). And VPA could almost entirely compensate this effect (0.8 fold). We therefore hypothesized that VPA has an impact on pDNA methylation in the context of TGE.

2011

Characterization of the 5HT3 receptor in live cells and in vitro

Veronica Ponce de Leon

The serotonin type 3 receptor (5HT3R) is a homopentameric, cation selective channel that mediates fast excitatory transmission in the nervous system. It is a member of the Cys-loop family of receptors that include the nicotinic acetylcholine receptor (nAChR), the γ-amino butyric acid (GABA) type A and C and glycine receptors. The 5HT3 receptor is involved in behavioral and digestive disorders and it is therefore an important therapeutic target. The 5-HT3 receptor was expressed in mammalian HEK cells (chapter 2) stably and transiently, and its expression was optimized to obtain 1.6 x 106 receptors per cell for its solubilisation (chapter 3) and purification (chapter 4). Solubilisation of the receptor was evaluated under different conditions such as using CHAPS and C12E9 detergents and homogenization procedures such as vortexing and thurraxing the membranes. Radioligand binding assays indicated that the amount of solubilised receptor was similar in both cases. The stability of the receptor at different temperatures was evaluated. The receptor was more stable when kept at -20 °C compared to 4 or 37 °C. Purification of the 5-HT3 receptor from HEK cells was performed by tandem IMAC (Immobilized Metal ion Affinity chromatography)/IMAC purification and IMAC/streptactin flow column chromatography. The 5-HT3R was obtained 50% pure and a yield of 10 pmoles of receptor binding sites per gram of starting material (dry cells) was obtained. Purified receptor preparations were homogenous. Finally, the receptor was concentrated 5.5 times, which allowed the incorporation of the receptor in lipid vesicles for further studies. Detergent solubilised 5-HT3R was incorporated in lipid vesicles and the functionality of the receptor was assessed by studying calcium ion permeability of the channel using calcium ion sensitive fluorophores (chapter 5). The effect of the cAMP dependent kinase (PKA), on the 5-HT3R was investigated by measuring the KD of the radioactive ligand [3H]GR65630 to the receptor in cells where PKA was activated or inhibited (chapter 6). Finally a dose-response curve of the binding activity of the 5-HT3R upon activation of the PKA was assayed in both HEK and CHO cells (chapter 6). Radioligand binding assays indicated that upon activation of the PKA, there was a decrease of the KD of 30% indicating an increase in the receptor binding affinity to 5-HT upon activation of the PKA (chapter 6). A kinase and cytoskeleton binding protein PDLI1 that was co-purified with the receptor was found phosphorylated upon PK activation (chapter 6). As PDLI1-like proteins are adaptor proteins between kinases, cytoskeleton proteins and sometimes receptors, the latter may be involved in the kinase activation of the receptor. These results contribute to the optimization of the receptor expression, solubilisation and purification in mammalian cells for its incorporation in lipid vesicles and for further structural studies. In addition, this work gave new insights to the mechanisms of channel regulation by PKA phosphorylation at the cell membrane and the intermediate proteins that are involved.

EPFL2009

Transient Gene Expression in HEK-293E Cells for Recombinant Protein Production

Sophie Nallet

The growing demand of biopharmaceutical products is boosting the market for therapeutic recombinant proteins (r-proteins). More than half of the 140 r-proteins that have gained approval for human therapeutic use are manufactured in mammalian cells that make possible complex post-translational modifications, like glycosylation. In the industry, r-proteins are manufactured by stable gene expression (SGE) following Good Manufacturing Practice (GMP) standards. Transient gene expression (TGE) in mammalian cells is an alternative method for the expression of r-proteins whose main advantages are rapidity and flexibility. TGE involves the expression of the protein of interest from plasmids, which are transfected into the cells with a non-viral DNA transfecting agent, usually polyethylenimine (PEI). DNA is transcribed from the plasmids without the latter having been integrated into the host genome. Despite the recent improvements in r-protein titers by TGE and the scale up of this method to the 100-L scale, TGE remains so far a tool for protein production for research purposes. Therefore, to determine if TGE can be used for manufacturing therapeutic r-protein following GMP standards, different features related to reproducibility and product quality from TGE of a recombinant anti-rhesus D immunoglobulin G in HEK-293E cells using linear PEI were characterized. To test the effects of the size distribution and chemical structure of PEI on TGE, different commercially available PEIs were characterized and their impact on transfection and r-protein expression were assessed. The results showed that both the molecular weight distribution and the chemical structure of the PEI had an effect on TGE. However, the small variations in size distribution and structure between the batches of PEI from the same supplier did not affect significantly the transfection and r-protein production by TGE. Then, the fate of PEI and plasmid DNA in cell culture over time and their removal from the final r-protein during purification were determined and measured. Most of the PEI and pDNA remained in the cell culture medium after transfection. However, both PEI and plasmid DNA, which are additional components in TGE compared to processes based on recombinant cell lines, could be reduced to a concentration below the limit of detection of the assays after Protein A affinity purification of the antibody. To test the reproducibility of a TGE process, 10 batches of transfected cells were run in 500-mL orbitally shaken bottles. The cell specific productivity of the antibody was 20.2 ± 2.6 pg.cell-1.day-1 on average for the 10 batches. The purity and size consistency of the recombinant antibody produced in those 10 batches was demonstrated by gel electrophoresis and size exclusion chromatography. Then, the glycosylation profile of the antibody produced by TGE in HEK-293E cells was determined by mass spectrometry and was reproducible over the 10 batches. Moreover, it was similar to the glycosylation profile of the antibody produced by 3 stable HEK-293E cell lines. Finally, as a proof of concept, TGE was applied for the development of a vaccine against the respiratory syncytial virus. The fusion protein of the respiratory syncytial virus was produced by TGE and used as an antigen for the development of a recombinant vaccine. TGE enabled the rapid evaluation of the candidate vaccine in animal trials. Overall, the results of this study suggest that TGE is a reliable and reproducible method for manufacturing r-proteins of a consistent quality, provided that some particular features, such as reagents quality and process parameters, are well defined and controlled. Since TGE makes possible the rapid production of virtually any protein, even toxic, it could be used to provide GMP approved biopharmaceuticals for clinical trials in a short time, reducing development costs of biological therapeutic products.

EPFL2010