The overall goal of this procedure is to express mammalian glycoproteins with appropriate mammalian glycosylation by targeting recombinant proteins to the ER and GOGI mediad secretory pathway. This method can help answer key questions in immunology such as what is the role of a specification, patterns in the structure and immune activation potential of antibodies and antibody fragments. The main advantage of this technique is that it uses human cells to express human proteins, which provides the appropriate cellular missionary to properly fold and modify the target proteins.
The incubator shaker for cell establishment should be operated at 135 RPM, 80%humidity, 8%carbon dioxide, and 37 degrees Celsius at least one hour prior to culture inoculation. Turn on the UV lamp of the biosafety cabinet, prewarm the sealed medium A and medium B bottles in a 37 degree Celsius water bath. Turn off the UV lights and sterilize the biosafety cabinet with a 70%ethanol and water solution.
Sterilize a 125 milliliter erlenmeyer growth flask with vented cap pipettes, pipetters, and prewarm media bottles by spraying with a 70%ethanol in water solution. Place these items in the biosafety cabinet throughout this procedure. All items must be sterilized in this manner before they are brought into the biosafety cabinet.
Prepare fresh culture medium for a 30 milliliter culture by withdrawing 26 milliliters of medium A and three milliliters of medium B and transferring into the 125 milliliter erlenmeyer flask. Mix by gentle shaking, warm, a vial of HEK 2 93 F cells previously frozen at minus 80 degrees Celsius in a water bath at 37 degrees Celsius to partially thaw the cells. This takes about one minute and the cells should not be completely thawed.
Next, sterilize the outside of the vial with the 70%ethanol in water solution and move it into the biosafety cabinet. Using a one milliliter pipette, withdraw the cell suspension and transfer it into the 125 milliliter erlenmeyer flask containing 29 milliliters of fresh culture medium. Close the cover of the culture flask and place the flask into the incubator shaker for 24 hours.
24 hours after culture inoculation. Check the cell density, sterilize the culture flask with the 70%ethanol and water solution and to move it into the biosafety cabinet. Using a one milliliter pipette and pipetter slowly withdraw 100 microliters of the culture and transfer it into a sterile 0.5 milliliter einor tube.
Close the cover of the culture flask and return the culture flask to the incubator shaker as soon as possible. Mix 7.5 microliters of a trian blue solution with 7.5 microliters of cells. Mix vigorously and then transfer 7.5 microliters of the mixture to a counting slide.
Turn on the automatic cell counter and place the counting slide into the loading chamber. The auto reader is activated and cells are counted automatically in the units of number of cells per milliliter and percent viability. Determine the volume of the donor culture required for transfer to a fresh culture medium to obtain a cell density of 300, 000 live cells per milliliter.
Prepare fresh culture medium as shown previously by transferring 23 milliliters of medium A and three milliliters of medium B to a fresh 125 milliliter culture flask. After that, remove the stock bottles of medium A and medium B from the biosafety cabinet to reduce the risk of their contamination. Retrieve the HEK 2 93 F cell culture flask from the incubator.
Spray it with the 70%ethanol and water solution and place it in the biosafety cabinet Using a new pipette. Withdraw the correct aliquot of cells from the culture and transfer it to the flask containing fresh culture.Medium. Transfer both cultures from the biosafety cabinet to the incubator shaker.
Grow the cells to an approximate density of two to 3 million live cells per milliliter prior to transfection. Check the cell density as demonstrated earlier and determine the amount of cells for the transfection. Using a sterile serological pipette.
Transfer the appropriate volume of cell suspension to a 250 milliliter centrifuge tube. Collect the cells by centrifugation at 100 times G for five minutes. After sterilization, transfer the tube containing the pelleted cells into the biosafety cabinet.
Use a sterile pipette to decant the supernatant consisting of spent. Culture medium vigorously resend the pelleted cells by pipetting up and down using 10 milliliters of fresh culture medium, and transfer to a prepared flask containing fresh transfection culture.Medium. Screw the cap on the vented cell culture flask and incubate the flask in the incubator shaker for 15 minutes to one hour while the cells are incubating.
Dilute the plasma DNA and POLYETHERAMINE or PEI stocks using fresh culture medium to a final concentration of 0.5 micrograms per microliter. Remove the culture flask with the dispersed cells from the incubator shaker and take it to the biosafety cabinet using a micro pipette and 300 microliters of plasma DNA to the culture and mix by gentle manual shaking at 900 microliters of PEI to the culture and mix again. Then at 3.8 milliliters of fresh culture medium.
Transfer the flask to the incubator shaker for 24 hours. 24 hours after transfection, the cell culture must be diluted. To prepare the dilution medium first, use a sterile one milliliter serological pipette to withdraw one milliliter of PREWARM 220 millimolar valproic acid or VPA and transfer it to a sterile 50 milliliter tube.
Next, use a sterile serological pipette to add five milliliters of prewarm medium B and 44 milliliters of prewarm medium A to the VPA solution. This results in 50 milliliters of fresh culture medium with a final concentration of 4.4 millimolar of VPA. Retrieve the transfected culture from the incubator.
Sterilize the exterior of the flask and place the flask in the biosafety cabinet. Add the prepared dilution medium to the culture. Transfer the flask back to the incubator shaker for an additional four to five days following transfection.
Monitor cell viability daily by the procedure demonstrated earlier. Aliquots should also be saved each day for protein expression analysis, five to six days after transfection or if the cell viability falls below 50%Harvest cells by centrifuging the cells plus medium at 1000 times G for five minutes. Collect the supernatant that will contain secreted protein at five milliliters of a 10%bleach solution to the HEK cell pellet and discard as a biohazard.
Subsequently, protein is purified from the collected supernatant. This expression system generated a high yield of glycosylated proteins illustrated by this typical expression pattern of IgG one fc after transient transfection of HK 2 93 F cells. Affinity purification using a protein column for IgG one FC or nickel column for histamine tagged GFP with FC gamma receptor three A resulted in proteins with high purity.
This system efficiently expressed isotopically enriched IgG one FC in this two dimensional NMR spectrum that correlates the resonance frequency of hydrogen nuclei and the directly bonded amide nitrogen. 15 atoms well dispersed signals were seen. Different glyco forms were produced with HEK 2 93 F and HEK 2 93 s cells under different culture conditions as shown by matrix assisted laser DESORPTION ionization mass spectrometry analyses.
IgG one FC expressed from HEK 2 93 s cells, harbored and glycans that were of a form with five anos plus two N acetyl glucosamine residues that contained no FU glucose glycans from HEK 2 93 F express material were complex type bi itinerary forms with core FU glucose and mostly having terminal N acetyl glucosamine or a small proportion of mono or dilated forms. The addition of an inhibitor of glycan ation to an expression conducted using HEK 2 93 F cells showed a greater than 90%reduction in fuco incorporation Once mastered, establishing the transient transfection can be completed in 1.5 hours on day one and 15 minutes on day two. After an expression period of four to six days, the solution containing the protein can be harvested in 15 minutes.
Finally, purification of the protein will take about 1.5 hours while attempting this procedure, it's important to remember to maintain a stable culture prior to transfection transfect actively growing cells and add DNA prior to adding PEI. Following this procedure. Other methods like protein functional characterization using a range of biophysical techniques can be performed in order to probe the structure and function of the expressed glycoprotein after its development.
This technique paved the way for researchers in the field of immunology to explore the structure activity relationship of IgG and glycosylation in vitro and in vivo. After watching this video, you should have a good understanding of how to prepare and purify glycoproteins using our combination of a K 2 93 F cells and the PZ N two vector.
