In this video, we describe a time and cost efficient procedure for expression of secreted proteins from made Durant HEC 2 9 3 T cells. Initially, a 40%Cofluent HEC 2 9 3 T cell culture is prepared in a six world plate. A transfection mixture is then produced by diluting gene juice, then test plasmids in serum free medium.
The transfection mixture is deposited on the cells, which are then incubated at 37 degrees Celsius to allow for protein expression. Detection of protein in the supan agent can be performed by western blot. Once a suitable construct is identified, the procedure can be scaled up using 10 layer cell factories.
Cells are transfected using PEI as the transfection. Reagent and protein can be harvested from the culture supernatants using tangential flow filtration followed by affinity chromatography. Ultimately, this platform allows expression of a wide range of macromolecules in milligram quantities.
The main advantage of this technique over existing methods such as balo virus based insect cell expression, is that large number of constructs can be screened and scaled up for expression with results obtained in a matter of three weeks. Demonstrating this procedure will be Hello Aiden for Shada Zini and Jonathan Cook. Three graduate students from my laboratory Begin by seeding 250, 000 HEC 2 9 3 T cells per well in a volume of one milliliter in a six well plate add one milliliter per well of 10%FBS pentre supplemented DMEM and S swir the plate to disperse the cells evenly.
Then incubate the cells overnight in a humidified 5%carbon dioxide incubator at 37 degrees Celsius. Once the cells reach a co fluency of about 40%replace the SUP natum with two milliliters of fresh medium to prepare the transfection mixture. First aliquot 90 microliters of serum free DMEM in an eend orph tube.
Then add three microliters of gene juice, the transfection reagent gently mix the contents of the tube and incubate it at room temperature for five minutes. Next, add 10 microliters of a 100 nanogram per microliter stock of the mini prep purified plasmid, DNA. To be transfected.
Mix the contents of the tube and let it sit at room temperature for 15 minutes. To perform the transfection, deposit the mixture onto the HEC 2 9 3 T cells. Drop by drop, swell the plate gently to distribute the transfection mixture evenly.
Then incubate the cells at 37 degrees Celsius. 24 hours later, add one milliliter of fresh medium to each well and incubate for a further 48 hours. Three days post transfection, harvest the supinate and detect the expression levels of the protein of interest by western blot.
Once an appropriate construct has been identified, the transfection procedure can be scaled up. Using 10 layer cell factories, fill a cell factory with 1.2 liters DMEM supplemented with 5%FBS. Then add 200 million HEC 2 9 3 T cells, distributing the cells evenly to all layers.
Four T 225 centimeter square cell culture flasks grown to 100%Co fluency contain about 200 million cells. Incubate the cells at 37 degrees Celsius overnight. Prepare the transfection mixture in a T 75 flask by diluting 840 micrograms of DNA in 84 milliliters of sterile one times.PBS.
Add 2.5 milliliters of one milligram per milliliter, polyethylene ion solution or PEI, and incubate the mixture at room temperature for 15 minutes. The solution should become cloudy. Next, slowly pour the transfection mixture into the cell factory and distribute it thoroughly.
Overall layers. Add valproic acid at a final concentration of four millimolar. This will increase expression yields once the transfection mixture has been added.
Incubate the culture at 37 degrees Celsius for four days to allow for protein expression. Harvest the medium four days post transfection and immediately clean the cell factory for reuse centrifuge the collected medium at 6, 000 G for 30 minutes at four degrees Celsius and filter it through a 0.22 micron steric cup vacuum filter apparatus to remove any particulate matter. Then use the centra mate tangential flow filtration system to concentrate the supernatant to 75 milliliters.
Once the supernatant is concentrated, the protein of interest can be purified by affinity chromatography. In this experiment, we purified the HA tached subunits of the human AAL two receptors extracellular domain using the large scale system described here, followed by high affinity anti THA chromatography. As shown in this kumasi stained SDS page analysis.
The extracellular domains of the interleukin two receptor alpha and gamma subunits migrate at the molecular weights of 40 kilodaltons and 46 kilodaltons. The heterogeneity of the EnLink glycans present on IL two R alpha and IL two R gamma caused band broadening on the SDS page gel. The higher molecular weight band represents contaminating BSA.
After watching this video, you should have a good understanding of how to express proteins from human cell culture using 10 layer cell factories. Do not forget that HC 2 93 T cells are considered biohazardous and should be handled at biosafety level two. Please wear proper personal protective clothing always, and surfaces should be disinfected according to institutional and governmental guidelines.
