TITLE: Purification of rat NLRP1, rat DPP9, and rat NLRP1-DPP9 complex from Sf9 Cells for structural and biochemical studies
AUTHORS: Louis R R Hollingsworth

[DESCRIPTION]
Protocol associated with "Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity" by Wang et al. and Saeij. Please address any questions to Bobby Hollingsworth (bobbyh11@vt.edu).

Protocol adapted from Huang* and Zhang* et al., Zhong and Chai 2021 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081665/)

[STEPS]
SECTION: Bacmid production
1. Obtain plasmids by emailing Dr. Jeroen Saeij (jeroensaeij@gmail.com) or from Addgene (deposition details will be added as a comment once processed). These include:
Lewis rat NLRP1 (Lew-rNLRP1) allele (allele 5 Uniprot ID D9I2G4) in pFastBac (His-TEV-rNLRP1[Lew]-FLAG)
Proteolysis-deficient Lew-rNLRP1 His-TEV-rNLRP1[Lew, S969A]-FLAG
Brown Norway rat NLRP1 (BN-rNLRP1) allele (allele 1, Uniprot ID D9I2F9) in pFastBac (His-TEV-rNLRP1[BN]-FLAG)
Proteolysis-deficient BN-rNLRP1 (His-TEV-rNLRP1[BN, S969A]-FLAG)
Rat DPP9 (rDPP9, Uniprot ID M0R781) in pFastBac (His-TEV-rDPP9)
Catalytically-deficient rDPP9 (His-TEV-rDPP9[S729A])
SECTION: Bacmid production
4. Transform ~100 ng plasmid into ~50 uL of DH10Bac cells. Proceed w/ normal transformation protocol, but incubate for 4 hours shaking at 37 before plating instead of the normal hour. This allows recombination and production of resistance proteins.

Helpful general transformation protocol:
https://www.addgene.org/protocols/bacterial-transformation/?gclid=Cj0KCQiA6t6ABhDMARIsAONIYyxEfw6FVU1ul6ajQQhP8_e4XHtCvMgIbuIPH4HqW6BKIascmX9YjJ4aAkRDEALw_wcB
 

SECTION: Bacmid production
3. Pour (or buy) bacmid LB agar resistance plates prior to proceeding. These are LB agar plates with 50 µg/ml kanamycin, 7 µg/ml gentamicin, 10 µg/ml tetracycline, 100 µg/ml Bluo-gal, and 40 µg/ml IPTG. Cover in foil (because plates contain doxycycline) and store at 4 degrees until needed. These generally last less than 1 month--blue/white discrimination becomes impossible if plates are kept too long.


SECTION: Bacmid production
2. Acquire other reagents and read up on the Bac-to-Bac system prior to continuing.

https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fbrochures%2F710_01985_BactoBac_bro.pdf&title=QmFjLXRvLUJhYw==https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fbrochures%2F710_01985_BactoBac_bro.pdf&title=QmFjLXRvLUJhYw==

http://kirschner.med.harvard.edu/files/protocols/Invitrogen_bactobacexpression.pdf
SECTION: Bacmid production
5. Plate all cells from an individual transformation onto bacmid plate(s) from step 3.
SECTION: Bacmid production
6. Wrap the bacmid plate in foil (because plates contain doxycycline) and keep in a 37 degree plate incubator for 2 days.
SECTION: Bacmid production
7. Pick at least 2 completely white colonies per construct and 1 negative control blue construct and grow in 5 mL of LB  containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline overnight. 
 

SECTION: Bacmid production
8. Miniprep the bacmid. I generally use this protocol from Dr. Owen Pornillos' lab (UVA):

  

 


SECTION: Bacmid production
9. PCR validate the insert on the bacmid. I've had limited success with Taq polymerases, so I generally do small (5-10 uL) Q5 polymerase reactions. Use M13 forward and reverse primers, which anneal to the backbone--there's a clear shift between the negative control (blue colony) and colonies that properly incorporated. With M13 primers, the PCR product should be ~2430 bp (HT vectors) + the size of the insert for a total of well over 5 kB.

 


SECTION: Bacmid production
10. Dilute the verified bacmid(s) to 1000 ng/uL in endotoxin-free/sterile TE buffer. Keep in the 4 degree short-term and freeze at -20 for long-term storage. Avoid freeze-thaw cycles.
SECTION: Virus production
11. Below, I detail a fairly generic an unoptimized protocol for generating virus. For maximum protein yield, careful consideration of virus MOI is required. This recent protocol from Dr. Mark Gorrell's lab also has some nifty tricks for optimizing sf9 expression:
https://doi.org/10.1016/j.pep.2021.105833

Plate 0.8 million cells from a suspension culture in a 6-well dish--1 well for each construct in addition to 1 well for the negative control. Use room-temperature complete SFX to bring the total volume between 1-2 mL in each well.

Wait 1 hr for cells to attach.60 min 

https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0007821_Cellfectin_II_Reagent_UG.pdf&title=VXNlciBHdWlkZTogQ2VsbGZlY3RpbiBJSSBSZWFnZW50
 

SECTION: Virus production
12. Meanwhile, heat up grace's insect cell medium (much like Opti-MEM for the mammalian-cell transfection savvy) to RT.
SECTION: Virus production
13. Add 100 uL grace's media to two microcentrifuge tubes (sterile) per construct.
SECTION: Virus production
14. Add 6 uL CellFectin II to one of the microcentrifuge tubes. 


SECTION: Virus production
15. Add 1000 ng of bacmid to the other 100 uL microcentrifuge tube.
SECTION: Virus production
16. Transfer the ~100 uL medium containing the bacmid to the tube containing CellFectin II. Pipette up and down gently, incubate 30 min.30 min 

SECTION: Virus production
17. Once Sf9s have attached (step 11), exchange medium with unsupplemented Grace's insect cell medium. Wash once with 2 mL of Grace's medium, remove, then add 1 mL of medium to keep on top of the cells. Return to incubator, or leave in the hood if step (16) is complete.
SECTION: Virus production
19. Add 800 uL Grace's medium to each microcentrifuge tube from step 16, bringing the total volume slightly above 1 mL. Quickly but gentle pipette up and down to mix and gently transfer to the sf9 well. Return the plate(s) to the 27 degree incubator for 4-5 h. 240 min 
 


SECTION: Virus production
18. Remove medium from sf9 plated cells, and immediately proceed to step 19
 

SECTION: Virus production
20. Remove medium from each well and add 2-mL complete sf9 medium (pre-warmed to room temperature). Return to the incubator and wait 3 days. 4320 min 
 


SECTION: Virus production
21. Plate 1.5 million cells from a suspension culture in a 6-well dish--1 well for each construct in addition to 1 well for the negative control. Use room-temperature complete SFX to bring the total volume between 1-2 mL in each well. Return the plate to the incubator for at least 1 h.60 min 

 

SECTION: Virus production
22. Harvest virus from the plate in step 20, which is in the cell culture supernatant. Transfer supernatant into labeled microcentrifuge tubes and spin at 2000g for 10 min to pellet cell debris. 2000 rpm, 10 min, 4 °C 


SECTION: Virus production
23. Transfer supernatant to fresh sterile microcentrifuge tube(s). This is considered the P1 virus (some call it P0, but for the sake of this protocol I will refer to it as P1).

 

SECTION: Virus production
24. Remove medium from the plate in step 21, and add 1 mL of fresh complete medium to each well. Transfer 700 uL - 1 mL of P1 virus-containing medium into each well. Return to incubator and wait 3 days.4320 min 

SECTION: Virus production
25. Harvest P2 virus as in steps 22 and 23.
SECTION: Virus production
26. Attach 15 million cells to a 15-cm plate, and allow them to attach for at least 1 h at 27 degrees. 
SECTION: Virus production
27. Add 1-mL of P2 virus to the 15 cm plate, and incubate for 3 days.4320 min 

SECTION: Virus production
28. Harvest P3 virus as in steps 22-23. Instead of a microcentrifuge tube, use falcon tubes, as there should be ~15 mL of virus-containing medium.
 

SECTION: Virus production
29. Add 5-mL of P3 virus to 200-mL suspension culture (at 2 million cells/mL). Store extra P3 virus-containing medium in the 4 degree. Wait 2 days.2880 min 

SECTION: Virus production
30. Harvest baculovirus-containing cells (BIICs). Centrifuge medium in sterile bottle(s) or several falcon tubes 1000 x g, 10 min 
 



SECTION: Virus production
32. Working in a cell culture hood, remove medium from bottles and resuspend BIICs in freezing medium. Aliquot resuspended BIICs into sterile microcentrifuge tubes (1 mL each). Label, and transfer them to -80 degree storage.
SECTION: Virus production
31. Prepare 20 mL freezing medium (at RT or 4 degrees) per construct. Freezing medium: Complete SFX medium with 10% DMSO and 10% FBS
SECTION: Protein expression
33. Grow suspension sf9 cells to 1 L at 2 million cells/mL. If cells more confluent, dilute down to 2 million cells/mL.
 

SECTION: Protein expression
34. Thaw an aliquot of frozen BIICs in your hand, and transfer to the insect cell flask to the under the cell culture hood. Return to the incubator for 2 d.

NOTE: Rat NLRP1 and DPP9 can be purified by themselves for biochemical studies. For the DPP9-NLRP1 complex, I co-express these to ensure that N-terminal degradation frees the C-terminal fragment for DPP9 sequestration.
SECTION: Protein expression
35. Harvest cells by centrifugation. Sterile technique is no longer necessary.2500 rpm, 20 min, 4 °C 

SECTION: Protein expression
36. Resuspend cell pellet(s) with ice-cold PBS and transfer them to 50 mL falcon tube(s) (or a different size if appropriate). This step is important, as it removes proteins and other components in the medium. Tubes can be split and aliquoted for individual protein preps, so budget accordingly.

Centrifugation 2000 rpm, 20 min, 4 °C 
SECTION: Protein expression
37. Gently remove PBS. 

Flash-freeze pellet(s) in liquid nitrogen and store at −80 degrees celsius. Alternatively, proceed directly to purification.
SECTION: Protein purification
38. Prepare and pre-chill the following buffers (NOTE: exclude TCEP for TRX-containing NLRP1 complexes, not discussed here):

Lysis buffer (100 mL, 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine abbreviated as TCEP, 5 mM imidazole)

Wash buffer (500 mL, 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM TCEP, 25 mM imidazole) 

Elution buffer (20 mL, 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM TCEP, 500 mM imidazole)

Size exclusion buffer (500 mL, 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP) 

 

SECTION: Protein purification
39. Thaw insect cell pellet and resuspend in 40 mL of lysis buffer (per L of cells).
 

SECTION: Protein purification
40. Transfer resuspended cells to a small metal beaker (glass works if you don't have one of these, just do not rest the sonicator against the bottom--this will shatter the beaker). Place beaker with cells in an ice-water bath and sonicate with a probe sonicator.

For a 1L pellet: 3 s on 5 s off, 3.5 min total on, 45% power, Branson Sonicator.
SECTION: Protein purification
41. Transfer to ultracentrifuge tubes and carefully balance the rotor.

 40,000 RPM for 1.5 h (45 Ti fixed-angle rotor, Beckman) or similar.  40000 rpm, 90 min, 4 °C 
SECTION: Protein purification
42. While centrifuging, pre-equilibrate Ni-NTA resin with lysis buffer (1 mL bed volume beads per 2L expression volume NLRP1) in cold lysis buffer.

CV = column volumes
SECTION: Protein purification
43. 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.
 

SECTION: Protein purification
44. Transfer Ni-NTA beads between all 50-mL falcon tube(s).
SECTION: Protein purification
45. Fill falcon tubes with cold lysis buffer to avoid bubbles while rocking. Nutate/gently rock in the cold room for 30 m.30 min 

SECTION: Protein purification
46. Centrifuge falcon tubes gently to pellet resin.

 800 x g, 5 min, 4 °C 
SECTION: Protein purification
47. Remove supernatant. Add 20 CV wash buffer and centrifuge again (batch wash).

 800 x g, 5 min, 4 °C 
SECTION: Protein purification
48. Remove supernatant. Add 5 CV wash buffer and transfer to a gravity column (we like Bio-Rad columns 7321010 and 7311550, depending on the bed volume).
SECTION: Protein purification
49. Wash the resin on-column with 25 CV ice-cold wash buffer. Be careful not to disturb the resin bed. 
SECTION: Protein purification
50. 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.

Incubate the resin (on the column) with 3 CV ice-cold elution buffer in the cold room. Gently mix, then wait 30 minutes. 30 min . Save buffer to rinse the column after elution to get as much protein off of the column as possible.
SECTION: Protein purification
51. Elute protein from the column and collect it in an Amicon Ultra 100 kDa cutoff spin concentrator (Millipore, UFC910096). Use ~1 CV buffer to wash the remaining protein off of the column after flow has stopped.
SECTION: Protein purification
52. Spin concentrate to ~0.5 mL.4000 rpm, 4 °C , transfer to a microcentrifuge tube.

SECTION: Protein purification
53. Spin microcentrifuge tubes hard (cold) to pellet any aggregated protein. 10000 rpm, 10 min, 4 °C 
SECTION: Protein purification
54. For apo proteins, separate them from aggregates and contaminants on a Superdex 200 increase 10/300 GL size exclusion column (Cytiva). Use size exclusion buffer as the running buffer. 

NOTE: A superose 6 increase 10/300 column is more appropriate for the rDPP9:rNLRP1 complex
SECTION: Protein purification
55. 
 
Collect peak fractions (annotated A14-B15 here for NLRP1 alone), run a quality control SDS-PAGE gel, concentrate, aliquot, and freeze as necessary.

 

Protein activity (or lack thereof for catalytic dead mutants) should be validated with a GP-AMC assay (see various DPPIV and DPP8/9 publications).