Protein Expression Eukaryotic cells - CHO Atf4

Protein expression refers to the techniques in which a protein of interest is synthesized, modified or regulated in cells. The blueprints for proteins are stored in DNA which is then transcribed to produce messenger RNA (mRNA). mRNA is then translated into protein. In prokaryotes, this process of mRNA translation occurs simultaneously with mRNA transcription. In eukaryotes, these two processes occur at separate times and in separate cellular regions (transcription in nucleus and translation in the cytoplasm). Recombinant protein expression utilizes cellular machinery to generate proteins, instead of chemical synthesis of proteins as it is very complex. Proteins produced from such DNA templates are called recombinant proteins and DNA templates are simple to construct. Recombinant protein expression involves transfecting cells with a DNA vector that contains the template. The cultured cells can then transcribe and translate the desired protein. The cells can be lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic protein expression systems are widely used. The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield. These expression systems include mammalian, insect, yeast, bacterial, algal and cell-free. Each of these has pros and cons. Mammalian expression systems can be used for transient or stable expression, with ultra high-yield protein expression. However, high yields are only possible in suspension cultures and more demanding culture conditions. Insect cultures are the same as mammalian, except that they can be used as both static and suspension cultures. These cultures also have demanding culture conditions and may also be time-consuming. Yeast cultures can produce eukaryotic proteins and are scalable, with minimum culture requirements. Yeast cultures may require growth culture optimization. Bacterial cultures are simple, scalable and low cost, but these may require protein-specific optimization and are not suitable for all mammalian proteins. Algal cultures are optimized for robust selection and expression, but these are less developed than other host platforms. Cell-free systems are open, free of any unnatural compounds, fast and simple. This system is, however, not optimal for scaling up.

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Found 1 matching solution for this experiment

PL_MmAtf4

Helene Faustrup Kildegaard, The Novo Nordisk Foundation Center f

Upstream tips
Experiments were performed in pre-sterilized polystyrene 96-square System Duetz HDW-microplates (CR1496c, Enzyscreen, Haarlem, Netherlands) capped with autoclaved low-evaporation sandwich Duetz covers (CR1296a, Enzyscreen). Cells (250 µL culture per well) were incubated in a Multitron Cell humidified incubation shaker (Infors HT, Basel, Switzerland) at following conditions: 85% humidity, 37°C, 5% CO2, 225 rpm (250 rpm when incubating transfected cells to minimize cell clumping), 50 mm orbit.
Protocol tips
The day before transfection, anti-clumping agent was removed from cells maintained in shake flasks by two centrifugation-based (200g, room temperature [RT], 5 min) washing steps. Plasmids were diluted in OptiPROTM SFM in a V-bottomed 96-well microplate (Greiner Bio-One). One half of total DNA amount was plasmid encoding model protein and the other half was either a mock plasmid (PL_TGExpr) or plasmids encoding target genes. Subsequently, FreeStyleTM MAX Reagent was diluted in OptiPROTM SFM and immediately after mixed with diluted plasmid. After 5 minutes of complexation, plasmid:FreeStyleTM MAX reagent mix was transferred to wells in HDW-microplate containing 2.5x105 cells in 250 µL CD CHO supplemented with 8 mM L-Glutamine and 50 U/mL Penicillin-Streptomycin (Life Technologies).
Downstream tips
3 hours post-transfection, anti-clumping agent was added to all wells reaching a final concentration of 2 µL/mL. VCD and viability were measured just before transfection (t=0) and at the following time points posttransfection: 24 h (day 1), 48 h (day 2) and 72 h (day 3). Supernatant samples were obtained by transferring cell suspension to V-bottomed 96-well microplate (Greiner Bio-One) and centrifuging (500g, RT, 5 min) the plate. Supernatants were recovered and stored at -80°C. When supernatant samples were obtained on day 2 and day 3 post-transfection (and not only on day 3), 30 µL cell suspension was diluted with 60 µL complete medium (3-fold dilution) to minimize effects of changing total cell culture volume.
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