TITLE: Purification of the CARD8-DPP9 Complex from Expi293F Cells
AUTHORS: Louis R Hollingsworth

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol associated with "DPP9 sequesters the NLRP1 C-terminus to repress inflammasome activation" by Sharif*, Hollingsworth*, Griswold*, et al., Bachovchin, and Wu. Please address any questions to Bobby Hollingsworth (bobbyh11@vt.edu). </div><div class = "text-block">General Expi293F expression protocol adapted from Dr. Andrew Kruse's protocols.</div><div class = "text-block">Protocol is largely the same as, "Purification of the NLRP1-DPP9 Complex from Expi293F Cells", except for no ADP present in buffers.</div></div>

[STEPS]
?. [Protein Expression]
Grow Expi293F cells (ThermoFisher, A14527) in Expi293™ Expression Medium (ThermoFisher, A1435101) to 2-3 million cells/mL at the desired culture volume, such as 1L. Choose the appropriate flask for the desired volume of cell culture to ensure appropriate gas exchange--I use a 2L Fernbach flask for a 1L batch of cells. If cells are grown slightly above 3 million cells/mL (up to 6 million cells/mL), dilute to 3 million cells/mL using fresh medium day-of. Otherwise, dilute below 1.5 million cells/mL and try again another day. Put cells back in the incubator once prepared, for now.
Cells should be >95 % viable (e.g. with trypan blue, Thermofisher 15250061) and doubling every 24 h. There are many strategies to grow cells to the appropriate volume/density. For example, I often grow 500-750 mL of cells to ~5-6 million cells/mL, then dilute the appropriate amount of cells with fresh medium in a new flask.
Warning: Expressing CARD8 or the CARD8-DPP9 complex in sf9 insect cells leads to mostly autoproteolysis-deficient product.
?. [Protein Expression]
Warm Opti-MEM to RT. In the hood, transfer RT Opti-MEM  to a small sterile, autoclaved flask. For smaller culture volumes, falcon tubes can be used.
[per 1L cells]
?. [Protein Expression]
Transfer endotoxin-free DNA encoding His-TEV-DPP9 (0.3 mg/1L cells; Addgene #166791) and DNA encoding CARD8-TEV-GFP-FLAG (0.7 mg/1L cells; Addgene #169991) into the Opti-MEM flask. Swirl to mix. Plasmids are available on Addgene: https://www.addgene.org/Hao_Wu/
For autoproteolysis-deficient CARD8 that only binds DPP9's site A, use the following: Addgene #169992 (CARD8 S297A-TEV-GFP-FLAG)
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Add polyethylenimine (PEI,  at   or  at  ). Swirl to mix. Incubate in the hood 30-45 min  . See the following resource for preparing PEI: https://www.addgene.org/protocols/transfection/
[per 1L cells]
[per 1L cells]
?. [Protein Expression]
In the cell culture hood, slowly pour Opti-MEM-DNA-PEI mixture into the flask containing cells (from step 1) while gently shaking. Return flask to the incubator, and incubate 24 hr.
?. [Protein Expression]
While gently swirling the flask, slowly transfer sterile glucose (9 mL, 45%, Sigma G8769 or in-house preparation) and sterile-filtered valproic acid (VPA, 10 mL, 300 mM, prepared from Sigma P4543) into the flask with the transfected cells. This step boosts protein production. At this stage, you could also take a small sample of cells and check for fluorescence in the green channel. ~50% of the cells or more should be GFP-positive. Return to the shaker for 4 d. .
?. [Protein Expression]
Take a small sample of cells and check for fluorescence in the green channel--50% of the cells or more should be GFP-positive. If the cells are not green under a fluorescent microscope, something went wrong with transfection or the cells were contaminated. Harvest cells by centrifugation . Sterile technique is no longer required.
Centrifuge: 2000 33, 20 min, 4 10
?. [Protein Expression]
Resuspend cell pellet(s) with ice-cold PBS, combine and transfer all resuspended cells to 2-50 mL falcon tubes (or a different size if appropriate). This step is important, as it removes proteins and other components in complete medium. Tubes will be frozen in aliquots for individual protein preps, so budget accordingly.Centrifugation
Centrifuge: 2000 33, 20 min, 4 10
?. [Protein Expression]
Gently remove PBS. Flash-freeze pellets in liquid nitrogen and store at −80 degrees celsius. Alternatively, proceed directly to purification.
?. [Protein Purification]
Prepare and pre-chill the following buffers:Lysis buffer (500 mL, 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP)On-column cleavage buffer (5.5 mL, 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, 0.2 mg TEV protease) MonoQ Low-Salt Buffer (250 mL, 150 mM NaCl, 25 mM Tris-HCl pH 8.0, 1 mM TCEP, filtered) MonoQ High-Salt Buffer (250 mL, 1 M NaCl, 25 mM Tris-HCl pH 8.0, 1 mM TCEP,  filtered) Dialysis Buffer (50 mL, 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP)
Buffers can be prepared ahead of time, but add TEV protease day-of to pre-chilled buffers. Ideally, do not keep reducing agent (+TCEP) buffer for more than 1 week. Buffers do not contain protease inhibitors because they might inhibit DPP9 and affect CARD8 binding. Work quickly, and always with cold buffers and/or on ice (unless otherwise specified)! TEV protease needs to be validated for high activity prior to use. Add more if necessary.All subsequent purification steps assume a pellet from 1L of cells. Scale accordingly.For purification of the VbP-bound complex, add 10 μM to all purification buffers.
?. [Protein Purification]
Thaw insect cell pellet and resuspend in 40-100 mL of lysis buffer (per L of cells).
I generally don't recommend using less than 40 mL of buffer for probe sonication, even with smaller expression volumes, such as from a 500 mL cell pellet.
?. [Protein Purification]
Transfer resuspended cells to a small glass beaker. Place beaker with cells in an ice-water bath and sonicate with a probe sonicator.For a 1L pellet: 2 s on 8 s off, 3.5 min total on, 40% power, Branson Sonicator.Cell lysis can be checked for completion under a microscope.
?. [Protein Purification]
Transfer to ultracentrifuge tubes and carefully balance the rotor. 40,000 RPM for 1 h (45 Ti fixed-angle rotor, Beckman) or similar.
Centrifuge: 40000 33, 60 min, 4 10
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While centrifuging, pre-equilibrate M2 flag beads with lysis buffer (1 mL bed volume beads per 1L expression volume) in cold lysis buffer.CV = column volumes
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Carefully remove the supernatant from the ultracentrifuge tubes and transfer to 50-mL falcon tube(s) or another appropriate vessel. Caution: handle delicately as to not disturb the pellet or lipid layer! Avoid 1) Cloudy liquid at the top of the tube, which contains lipids and other junk, and 2) the cell pellet.Keep the cell pellet on ice just in case lysis was incomplete.
?. [Protein Purification]
Transfer M2 flag beads between all 50-mL falcon tube(s).
?. [Protein Purification]
Fill falcon tubes with cold lysis buffer to avoid bubbles while rocking. Nutate/gently rock in the cold room for 4 h. .
4 °C
Do not stop here and leave the protein overnight. It might aggregate and it's prone to nonspecific protease digestion. CARD8 does behave much better than NLRP1, however.
?. [Protein Purification]
Wash a MonoQ column with 10 mL water, 10 mL high salt buffer (pump B), then 10 mL low salt buffer (pump A) to equilibrate it for later. Keep column and buffers refrigerated.
?. [Protein Purification]
Centrifuge falcon tubes gently to pellet resin.
Centrifuge: 800 34, 5 min, 4 10
?. [Protein Purification]
Remove supernatant. Add 20 CV lysis buffer and centrifuge again.
Centrifuge: 800 34, 5 min, 4 10
The flag beads should be very green at this stage or there were expression issues.
?. [Protein Purification]
Remove supernatant. Add 5 CV lysis buffer and transfer to a gravity column (we like Bio-Rad columns 7321010 and 7311550, depending on the bed volume).
?. [Protein Purification]
Wash the resin on-column with 25 CV ice-cold lysis buffer. Be careful not to disturb the resin bed.
?. [Protein Purification]
Let the remainder on top of the resin bed drain out, but do not let the bed dry. Stop-up the column as soon as it stops flowing.Cleave off the TEV-GFP-FLAG tag by incubating the resin with 5-CV (5 mL) on-column cleavage buffer as soon as the column is stopped up. Gently agitate the resin within the column then let stand. 1 h @ RT  . Save a little buffer to rinse the column after elution to get as much protein off of the column as possible.
You should add the freshly-thawed TEV protease to the buffer immediately before on-column cleavage. The resin will stay green (TEV-GFP-FLAG left behind). I recommend mixing everything, taking a 10-uL sample, and immediately quenching the recovered sample with SDS-PAGE buffer. You can mix and take samples in this fashion every ~15 minutes. Run out the full protein time course to on an SDS-PAGE gel to confirm complete cleavage by ~1 h, and adjust amount of TEV or the cleavage time in future protein preparations.
?. [Protein Purification]
Elute from the column and collect in 1.5 mL microcentrifuge tubes. Use the remaining ~0.5 mL buffer to wash the remaining protein off of the column.
?. [Protein Purification]
Spin microcentrifuge tubes hard (cold) to pellet any aggregated protein.
Centrifuge: 10000 33, 10 min, 4 10
?. [Protein Purification]
Take a 10 uL sample and add to 4X SDS sample buffer for an SDS-PAGE gel at a later time.
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Load all protein onto a MonoQ 5/50 GL column and begin anion exchange. Use a 5-mL loop, or inject multiple times with smaller loops.Program: 5 mL load, 5 CV wash, 0 to 100% pump B (high salt buffer) over 15 CV, final wash with 5 CV. Collect 0.5 mL fractions.
?. [Protein Purification]
Take the fractions with major peaks and run an SDS-PAGE to confirm protein identity.
?. [Protein Purification]
Measure the concentration (nanodrop A280) of fractions of note and record. Concentrate or dilute desired fractions. For cryo-EM, 0.3-0.5 mg/mL (assuming ε=1);  works well.
?. [Protein Purification]
Dialyze protein into Dialysis Buffer using a Slide-a-lyzer (ThermoFisher 66384). NOTE: this is only necessary if crosslinking is required for downstream applications, e.g. for cryo-EM.
This requires a buffer change after 2 hr followed by further dialysis (4 h to overnight). However, it's usually late at this point so I generally dialyze overnight and change the buffer in the am. I'll give it 4 h following buffer change before making cryo-EM grids or using the protein for other purposes. Be careful with the buffer exchange, particularly when handling the membrane.
?. [In vitro assays]
Carefully remove dialyzed protein, proceed with downstream application.