Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The overall aim of the following video is to demonstrate a simple and affordable technique for producing a good yield of protein or protein complexes using a mammalian expression system. This is achieved by culturing HEC 2 93 F cells in suspension in a humidified shaking incubator with an atmosphere maintained at 5%carbon dioxide. As a second step, the cells are transiently transfected with one or more high copy number expression plasmids.

Using the transfection agent polyethylene EIN or PEI, the cells were allowed to grow for 48 hours before harvesting. Next protein purification is performed using affinity resin, followed by gel filtration to purify the protein complex of interest in order to carry out structural and functional studies. Ultimately, this affordable and simple mammalian expression system enables expression and purification of proteins and protein complexes that are difficult to express in bacterial cells.

The Main advantage of this approach is its convenience and versatility for the expression of protein complexes. It is almost as easy as making proteins in bacteria, especially since you can mix and match protein expression vectors. The method can initially be used in small scale to test the expression of protein compresses, but it's readily scalable so as to produce good yield of protein compresses For structural and functional studies, The key to success is maintaining cells in a healthy state, free from bacteria or yeast infection.

As antibiotics in the media greatly reduce your protein yield. To begin, remove a cryo vial containing one milliliter of HEC 2 9 3 F cells at a density of 20 million cells per milliliter from the liquid nitrogen and quickly thaw in a 37 degrees Celsius water bath. Break up any clumps by pipetting up and down a few times and pipette the whole content into a 250 milliliter conical cell culture flask containing 34 milliliters of Prewarm media.

Place the culture in a humidified orbital shaking incubator at 37 degrees Celsius, 120 RPM and 5%carbon dioxide after 48 hours. Check cell viability by trian blue. Staining normal cell viability at this stage is approximately 70%when the cells will have reached a count of approximately 1 million cells per milliliter.

Massage them to a final count of 0.35 million cells per milliliter into a 250 milliliter conical cell culture flask containing 60 milliliters of prewarm media continue to massage the cells every 48 hours or when they reach account of approximately 2 million cells per milliliter. Shown here is a sample of Trian blue stain HEC 2 9 3 F cells at 2.3 million cells per milliliter from a stock ready to be seeded for transfection cells should only be present as single or dividing cells. If cells are overgrown, they will form large clumps and will have a very low viability as shown in this picture.

If cells are forming clusters, these may be broken up by vigorous vortexing for 25 seconds. To begin the large scale cultures seed the cells at half a million cells per milliliter into a final volume of 300 milliliters in each one liter roller bottle. Incubate for 24 hours in a humidified orbital shaker at 37 degrees Celsius, 135 RPM and 5%carbon dioxide until cells reach a density of 1 million cells per milliliter.

Next pipette a total of 300 micrograms of filter sterilized DNA into 30 milliliters of phosphate buffered saline or PBS and vortex vigorously for three seconds. Then add 1.2 milliliters of 0.5 milligrams per milliliter. Filter sterilized PEI to the PB SDNA solution and vortex vigorously for an additional three seconds.

Incubate the mix at room temperature for 20 minutes before adding the mixture to the cells following cot. Transfection incubate the cells in a humidified orbital shaking incubator for further 48 hours at 37 degrees Celsius, 135 RPM and 5%carbon dioxide after 48 hours. Harvest intracellular proteins by centrifuging cells at 3000 times G for five minutes.

If the cell pellet is not for immediate use, it can be stored at minus 80 degrees celsius. This protocol is optimized for the purification of protein complexes from the nucleus in which one of the proteins has a flag tag. To begin purification resus, suspend the cell palette into approximately 40 milliliters of pre chilled lysis, buffer per liter of culture, using a combination of pipetting up and down several times, and a glass homogenizer sonicate for three cycles of 15 seconds on 15 seconds off.

Then centrifuge your 108, 000 times G for 25 minutes at four degrees Celsius and retain the supine natant. Next equilibrate 1.2 milliliters of Anti-Flag packed aros resin per liter of culture by washing three times with resin equilibration buffer incubate the supinate, which represents the whole cell extract with the affinity resin in one or more 50 milliliter centrifuge tubes gently rotating for 30 to 120 minutes of four degrees Celsius following incubation centrifuge at 3000 times G for one minute at four degrees Celsius. After discarding the supinate, transfer the resin into a 15 milliliter centrifuge tube and wash with a total of 45 milliliters of pre chilled buffer, one centrifuges before and then discard the supinate repeat washing and centrifugation using high salt buffer, followed by low salt buffer and TEV cleavage buffer.

Ensure each wash is sufficient to fully resuspend the resin but no longer. Next, collect a 10 microliter sample of resin and dilute into one volume of two times protein loading buffer for analysis to represent the bound protein sample. Do not use reducing agents since they will release the antibody from the resin.

Then resuspend the resin into 10 milliliters of pre chilled TEV cleavage buffer at approximately 40 micrograms of TEV protease per liter of original culture and mixed by inverting the tube several times. Levels of TEV protease can be optimized according to the amount of express protein. Replace the tube atmosphere with 100%nitrogen gas to prevent protein oxidation before gently rotating overnight at four degrees Celsius the next day.

Centrifuge the sample at 3000 times G for 10 minutes, then transfer the supinate into an ultra centrifugal filter with an appropriate molecular weight cutoff and concentrate down to 500 microliters. Collect a 10 microliter sample of the concentrated protein and dilute into one volume of two times protein loading buffer for analysis. This sample represents the TEV eluate sample.

Then collect a 10 microliter sample of the resin and dilute into one volume of two times protein loading buffer. This sample represents the post TEV resin sample. The next step is to run the protein through a size exclusion column and appropriate gel filtration matrix should be used according to the size of the complex of interest.

In this case, we have used a 10 by 300 millimeter S 200 size exclusion chromatography column A equate the size exclusion chromatography column with gel filtration buffer. Filter the protein through a 0.22 micron filter and load the sample into the column. Proceed to collect the fractions as they come off the column.

As a final step, perform SDS poly acrylamide gel electropheresis or SDS page on the collected samples, as well as the gel filtration fractions, histone DS SLA one or HD one suppressor of defective silencing three or SDS three and syn three, A HDAC interaction domain or syn three, A HID form a stable turny complex. The affinity purification of the turny complex can be observed through SDS page. Shown here is the purified complex produced by cot, transecting two liters of cells.

The bound protein lane shows the complex bound to the affinity resin. Following TEV digestion, the tag present on sin three A HID is cleaved off and the complex is alluded from. The resin shown here is a chromatogram of the protein complex purified using a 10 by 300 millimeter S 200 size exclusion chromatography column.

Note that the pure complex eludes close to the void volume because it forms a dimer in solution. Finally, SDS page analysis reveals the purified protein in the gel filtration fractions. After watching this video, you should have a good understanding of how to grow, maintain, and cotran suspension grown HEC two and three app cells, as well as how to affinity, purify, and concentrate your protein.

Complex Healthy cells will be essential for the success of experiments, so make sure that they are grown and maintain in their conditions and passage regularly. Don't be put off by tissue culture. It's easy for structural biologists to express and purify large amounts of protein and protein complexes from mammalian cells.