#HEK293 cells are a host of choice for many biopharmaceutical companies and thus also a centerpiece of ongoing developmental research. Lets look at an easy HEK-F transient expression protocol for protein production in Erlenmeyer flasks.
Protocol:
Day -1: Maintain HEK-F cells in #Freestyle F-17 media supplemented with 4mM L-Glutamine and 0.1% #Pluronic F-68. Conditions are 37C , 5% CO2 and 25mm orbital shaking diameter in an #Erlenmeyer flask.
Day 0: 1. Count the cells and maintain to 2 million cells/ mL if required.
2. Prepare a fresh stock of #PEIMax (PEI MAX® - Transfection Grade Linear Polyethylenimine Hydrochloride, MW 40,000 from PolySciences) by dissolving appropriate amount in endotoxin-free water at 1mg/mL concentration. Maintain the pH to ~7 using 5M #NaOH and filter sterilize the solution.
3. Dilute plasmid #DNA to the concentration of 1ug/mL in 10% of final volume in Freestyle F-17 media.
4. Dilute PEI Max to the concentration of 3ug/ mL in 10% of final volume in Freestyle F-17 media.
5. Add diluted PEI Max to the diluted #DNA, mix gently by swirling and incubate the mixture for 15-20 minutes at room temperature, undisturbed for complex formation.
6. After incubation, add the complex to the culture gently with pipette while swirling the flask, and return the flask to the incubator.
Day 1: 1. Prepare the following Feeds:
2. Count the number of cells and ascertain viability of the culture.
3. If the culture is found healthy and growing, add the following to the culture: #Tryptone N1 to 0.5% of working concentration, Feed C to 5%, D-Glucose to 4g/L and L-Glutamine to 4mM of working concentration. Keep adding the feed every 48 hours until viability drops below 80%.
Harvest Day: 1. Spin the culture at 4000-6000 RPM for 30 mins at 4C.
2. For secretory protein, harvest the supernatant and add 0.02-0.03% sodium azide and Protease inhibitor, filter sterilize and proceed to purification.
3. For intracellular protein, wash the pellet with #PBS and proceed to purification.
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