The overall goal of this protocol is expression screening of multiple construct of protein arginine methyltransferases in large-scale production to obtain the milligram quantity of the protein for biochemical, biophysical, and structural studies in the medium supported, cost efficient mode in the coverage of expression platform S, baculovirus expression vector system. Dr.Alma Seitova will present the following steps. Dilute the exponentially growing SF9 cells to the final cell density of 0.4 million per mil in the serum-free insect media, and pour into the sterile reagent reservoir.
Use a programmable multi-channel pipette to seed 0.5 mils of the diluted SF9 cells to each well of the 24-well plate. This volume is enough to ensure even coverage of the working surface of the well. At the same time, it does not dilute the transfection mix too much, which enables transfection efficiency.
Label one well of the plate as cell only, and use it as a control for the comparison of the transfected and non-transfected cell to assess for potential signs of infection. Incubate the plates at 27 degrees until needed, approximately one hour, to let the cells attach to the cell culture plates. Dilute well-mixed transfection reagent in the unsupplemented insect medium and a sterile reagent reservoir, and mix gently for 10 seconds.
Using a 12-channel pipette, transfer 102 microliters of the diluted transaction reagent into a sterile 96 MicroWell plate. Transfer 10 microliters of 0.2 micrograms per microliter of recombinant bacmid DNA into the corresponding well of the 96-well MicroWell plate and mix by gently shaking, tapping the plate from the sides. Incubate the transfection mix for 15 to 20 minutes for a complex formation.
Using an adjustable six-channel pipette designed for the transfer between a 96 and 24-well plate, overlay the transfection mix onto the cells drop-wise in corresponding wells of the transfection plates, and incubate for four hours at 27 degrees. To ensure even distribution of the transfection mix over the cells'monolayer, gently rock the plates back and forth several times during the incubation time. Four to five hours after transfection time, add 1.5 mils of serum-free insect media.
Supplement it with 10%final of heat-inactivated fetal bovine serum and 1%antibiotic and antimycotic. Incubate cells in a 27-degree incubator for 72 to 96 hours. Gently rock the transfection plates once a day when possible.
Look for signs of infection evident in transfected cells at 72 to 96 hours post-transfection time. Four to five days after transfection, signs of infection should be evident in the transfected cells in comparison with the control cells under an inverted microscope, and the initial recombinant baculoviruses secreted into the cell culture medium should be ready to collect. Use a programmable electronic multichannel to allow for simultaneously the collection of P1 viruses, the infection of freshly seeded SF9 cells in the infection of suspension cells in their 24 well blocks with 150 microliters of the P1 viruses.
Spin down the rest of the collective P1 viral stocks for 15 minutes, cover the 24 well blocks with suspension culture of the infected SF9 cells with an AirPore sheet. Incubate the 24 well block at 27 degrees with shaking at 245 RPMs for 72 to 96 hours. Four days before scheduled production time, split exponentially growing SF9 cells, at a final cell density of 2 million per mil into Erlenmeyer glass shake floss with baffles in serum free insect media containing 1%final antibiotic and antimycotic.
This step will generate suspension culture of infected insect cells and P2 viruses in the supernatant for the infection of new cells in the production batch. Add the corresponding P2 viruses and incubate infected cells at the lower temperature of 25 degrees to slow cell growth, shaking at 165 RPMs on an orbital shaker with an one inch stroke. Four days before the scheduled production time, Seed two liters of exponentially growing SF9 cells in insect serum free media to the cell density of 1 million per mil, in the 2.8 liter Fernbach shake flask.
Shake the flask at 27 degrees with shaking at 150 RPMs. Research scientist Ashley Hutchinson will demonstrate the following protocol. Split four liters of exponentially growing SF9 cells and insect serum free media to the final cell density of 4 million per mil into the large five liter reagent bottles.
Add 10 to 12 mils of the suspension culture of the baculovirus infected insect cells. Incubate the infected culture of the SF9 cells in a shaker shaking out 145 RPM at a lower temperature of 25 degrees Celsius for 72 to 96 hours. Milligram quantities of several recombinant proteins from the PRMT family express in the baculovirus immediate production in SF9 cells are used for biochemical biophysical instructional studies, as can be seen in figure five.
At the SGC, crystal structures were solved in deposited into the protein data bank for the full length or truncated forms of the proteins PRMT4, 6, 7, and 9 with various chemical probe and inhibitors. Expression plasmids for these PRMTs were deposited to add gene collection by SGC and are available to the research community. In this video, we have emphasized key elements for successful expedition screening of their multiple construct of the protein arginine methyltransferases in the large-scale production in the baculovirus expression system.
Use of the regular adjustable and programmable multi-channel pipettes, made their whole process more rapid than efficient. Use of high-performance and less cell toxic for the cells, transfection reagent for the generation of recombinant viruses led to more cost-efficient and this cell handling transfection protocol. Infection of the large scale batch for protein production with suspension culture of baculovirus infected in six cells significantly reduced the labors in time consuming step in vital starter amplification.
Is for example, centrifuging this with special culture of infected cells. That's of course in excluded the extra handling of the infected cells in avoid in their tighter in virus degradation. Suspension culture culture cell maintenance in this scale up production in the culture vessel was high field volume help us to overcome limitation of the production volume and adopt a large scale platform.
This is highly useful for the labs with no access to the bioreactors or limited space in the production pipeline. Although our protocol have been described for the proteins of the protein arginine methyltransferases family, the same approach can be applied to any other proteins family requiring expression platform to obtain a sufficient quantity of their protein for biochemical biophysical instructional studies.
