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Jenkins Lab MHC Class II Tetramer Production Protocol – 01/08/16 check for updates at www.jenkinslab.umn.edu

This protocol describes in great pain and detail how to produce soluble fluorescent MHC class II tetramers containing a covalently linked peptide antigen. The design of the tetramer incorporates Fos-Jun leucine zipper motifs to force dimerize the coexpressed MHCII α and β chains (Teyton, et. al., J. Exp. Med. 183:2087), and the E.coli BirA signal sequence (Schatz, et. al., Protein Science 8:921) on the α chain to allow for site-specific biotinylation. The resulting biotinylated peptide:MHCII (pMHCII) heterodimers are tetramerized with fluorochrome-labeled streptavidin.

Recently, we have incorporated a "disulfide trap" strategy of stabilizing the peptide in the MHC binding pocket by introducing a cysteine residue into the linker sequence, which forms a disulfide bridge with another cysteine (α72C) introduced into a neighboring position in the α1 helix of the MHCIIα chain (Kappler, et. al., Proc Nat Acad Sci USA 107:10978). Note that this strategy is only feasible if cysteine is not a residue within the antigenic peptide sequence.

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I. Clone Expression Plasmids The covalently-linked peptide antigen is encoded by a fusion to the N-terminus of the MHCIIβ chain via a flexible glycine-serine linker. Design peptide antigens to include the 9-mer MHCII binding core plus two additional N-terminal residues. This sequence is flanked on the 5' side by a unique XmaI restriction site (within the peptide signal sequence) and on the 3' side by a unique SpeI restriction site (within the linker sequence). To clone a new peptide sequence, design a set of overlapping oligos encoding the new peptide sequence plus flanking sequences encompassing these restriction sites. For constructs not utilizing the disulfide trap strategy, simply substitute the cysteine (C) residue in the linker with another glycine (G). The following sequence is designed for an I-Ab β chain construct, but a similar strategy can be applied to other MHCIIβ constructs.

1) Design overlapping oligos for the insert.

The 5' end of the sense oligo should start 3 bases 5' of the XmaI site and extend 3' for about 70-80 bases. The 5' end of the antisense oligo should start 3 bases 5' of the SpeI site and extend 3' for about 70-80 bases. Request PAGE-purification of these oligos when ordering (requires 100 nmol scale production).

2) Anneal oligos slowly, preferably in a thermocycler. a) Make 100 µM stocks of each oligo in TLE (10 mM Tris pH8, 0.1 mM EDTA) or dH2O. b) Mix together 5 µl of each oligo in a 0.2 ml PCR tube. c) Run the following program in a thermocycler:

i) 95C 30" ii) step -0.1C/s to 4C iii) 4C forever

d) Keep on ice. 3) Run double-stranded product on 2% agarose gel and purify band of expected size using DNA

gel extraction kit (Qiagen 28704). http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf.

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4) Digest parent vector and new insert fragment with XmaI and SpeI restriction enzymes (New England Biolabs R0180S, R0174S). http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/default.asp

5) Run digested product on 2% agarose gel and purify band of expected size as in step 4. 6) Ligate purified digested vector and insert fragments (Invitrogen 15224).

http://www.invitrogen.com/content/sfs/manuals/t4dnaligase_1U_man.pdf 7) Transform XLI Blue, DH5α, or equivalent competent cells and plate out on LB agar

containing 100 µg/ml ampicillin (Stratagene 200249). http://stratagene.com/manuals/200249.pdf

8) Miniprep 4-8 colonies (Qiagen 27104). Spot backup colonies on new ampicillin plate for future maxipreps. http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QP_Miniprep/1043788_HB_QIAprep_122006.pdf

9) Screen minipreps by sequencing the insert region. Use "I-Ab β peptide" primer (5'-ATCATCTCAGTGCAACTAAA-3'). http://www.bmgc.umn.edu/bmgc/facilities/home.html

10) Maxiprep clone with correct sequence (Qiagen 12262). http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QIAfilter/1034638_HB_QIAfilter_112005.pdf

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II. Transfect Drosophila S2 Cells Expression of biotinylated pMHCII in Drosophila S2 cells is carried out in most part according to the DES Drosphila Expression System from Invitrogen (K5130-01). http://www.invitrogen.com/content/sfs/manuals/des_man.pdf In general, S2 cells are grown at 28C without CO2. Note that non-CO2 incubators tend to be very dry, so be vigilant about maintaining humidity in the incubator with a tray of water. S2 cells can be spun down at about 1400 rpm for 5 min in a tabletop centrifuge. A T25 flask of parental cells in 5 ml complete medium (see recipes) should be continuously maintained, split no more than 1:10 every 3-4 days. S2 cells require a soluble autocrine growth factor for optimal growth, so any split beyond 1:10 will result in a substantial slowdown in growth and maybe even death. Always warm media up to 28C in the incubator before adding to cells (do not use a water bath). To avoid contamination, pipet freshly made media into 40 ml aliquots in 50 ml tubes. Avoid tipping tubes so the cap stays dry. Optimal medium volumes for tissue culture vessels are:

T25 5 ml T75 15 ml T225 50 ml 1L shaker 500 ml 2L shaker 1 L

In vivo biotinylation of MHC is achieved by co-transfection of an expression plasmid for the E. coli BirA enzyme (see Yang, et. al., Human Immunology 65:692-699). The drug resistance gene is encoded on a separate plasmid and transfected at 1/9th the amount of each other gene. This limits the extent of cells gaining drug resistance without expressing the genes of interest. 1) Using healthy S2 cells in log growth phase, set up 3 x 106 cells in 3 ml of complete medium

(see recipes) in 60 mm tissue culture dishes. Use medium from Invitrogen (11720-034), as we have had suboptimal growth with media from other vendors. Incubate overnight at 28C. You can place three 60 mm dishes inside a 150 mm dish to provide an additional sterile barrier.

2) Transfect cells using the Calcium Phosphate Transfection Kit (Invitrogen K2780-01) included in the DES kit. A detailed protocol is provided in the DES product manual. http://www.invitrogen.com/content/sfs/manuals/des_man.pdf. Make sure all reagents are warmed to RT prior to transfection. a) For each transfection, set up and mix in 1.5 ml microfuge tube:

sterile dH2O 236 µl CaCl2 (2M) 240 mM 36 µl pRMHa-3 I-Ab alpha-C BirA plasmid (1 µg/µl) 9 µg 9 µl pRMHa-3 I-Ab beta (peptide)-C plasmid (1 µg/µl) 9 µg 9 µl p18 BirA plasmid (1 µg/µl) 9 µg 9 µl pCoBlast plasmid (1 µg/µl) 1 µg 1 µl 300 µl

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b) Slowly add contents dropwise over the course of 1 min to another 1.5 ml microfuge tube containing 300 µl 2xHEPES HBS while vortexing. This process produces the fine precipitate that mediates transfection of DNA.

c) Incubate mixtures at room temperature for 30-40 min. d) Slowly drop mixture into cell culture while swirling dish. e) Incubate 1 d at 28C. f) Replace medium with fresh complete medium.

i) Pipet medium from the dish into a 15 ml centrifuge tube. ii) Carefully rinse cells with 1 ml fresh medium; add/pool to 15 ml tube. iii) Add 1 ml fresh medium to dish so the cells don't dry out. iv) Spin down tube. v) Resuspend pellet with 2 ml fresh medium; transfer to dish.

g) Incubate 2 d at 28C. 3) Put cells into selection. Cells should always be kept in medium containing blasticidin after

this point, as they will lose expression over time without selective pressure. a) Repeat procedure described in step II.2.f, but use fresh complete medium containing 25

µg/ml blasticidin (Invivogen ant-bl-5b)(not Invitrogen). Add the blasticidin to the medium first before adding to cells.

b) Incubate 3 days at 28C. c) Gently resuspend the cells by pipetting and transfer 3 ml into 27 ml of SFM (see recipes)

+ blasticidin (25 µg/ml), then add 15 ml to two T75 flasks (1:10 split). d) Incubate at 28C. The goal at this point is to produce 2.5x108 total cells. This takes about 5

days. Remove a sample on day 5 and do a trypan blue count to see if the culture contains 2.5x108 total cells. If not, then continue until this number is achieved.

e) Add the 30 ml of SFM from the two T75 flasks containing ~2.5x108 cells to 470 ml of SFM + blasticidin in a 1 L shaker flask. Incubate at 28C with constant shaking at 125 rpm in a shaking incubator.

f) Incubate at 28C until cells reach ~5x106 cells/ml. This will take about 4 days. 4) Freeze some cells at this point.

a) Remove 5 ml of the 500 ml cell, spin down the cells, remove 2.5 ml before resuspending the pellet, then resuspend the pellet and add 2.5 ml of complete medium containing 20% DMSO.

b) Freeze down 5 x 1 ml aliquots. c) Thawing cells: see DES Drosphila Expression System from Invitrogen (K5130-01), page

5. 5) Induce the cells in the remaining 495 ml to produce biotinylated pMHCII monomers.

a) Add 0.4 ml of 1M CuSO4 and 1ml of 1 mg/ml D-biotin (Pierce 29129). b) Incubate 8 days at 28C, 125 rpm.

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III. Purify Soluble pMHC Soluble pMHCII monomers are enriched from cell culture supernatant via His-Bind chromatography targeting the 6x His epitope on the C-terminus of the MHCII beta chain. The nickel column eluate is then passed over a Pierce Monomeric Avidin UltraLink Resin (Thermmor Scientific, number 53146) and eluted with free biotin to purify biotinylated pMHCII molecules. A. Isolate cell culture supernatant. 1) Spin down induced S2 cells from step IV.6. (500 ml conical tubes at 2500 rpm for 10 min in

a Beckman tabletop centrifuge). Spin down supernatant again in new tubes. 2) Filter supernatant first through 3 µm pore-size Whatman paper (Whatman 1003-240), and

then directly through a 0.45 µm vacuum filter. Aggregated material in the supernatant can make this a slow process. Multiple filters may be needed.

3) Dilute 1:1 with ddH2O. 4) Add 0.1% azide and store at 4C. Sometimes aggregates will form in the filtered supernatant

as it is stored in the cold. If this happens, re-filter it. B. Perform nickel affinity chromatography. Nickel affinity chromatography is carried out using the Novagen His-Bind Purification Kit (Novagen-EMD Biosciences 70239-3). http://www.emdbiosciences.com/docs/docs/PROT/TB054.pdf It is important to use a chelating resin based on iminodiacetic acid (IDA) instead of nitriloacetic acid (NTA) reactive groups. This is because free copper ions in the cell culture supernatant will efficiently compete with His-tagged protein/ion complexes for binding to NTA groups, but not to IDA groups (see Lehr, et. al., Protein Expr Purif 19:362-368). 1) Add 4 ml of well mixed His-Bind nickel resin slurry to a 1 x 10 cm glass column (Kontes

420400-1010) clamped on a ringstand. The resin is supplied as a 50% suspension slurry, so 2 ml of packed resin will remain in the column after the liquid portion has drained away. This amount of resin should have a binding capacity of about 16 mg protein (8 mg per ml of resin).

2) Wash the column by adding 6 ml dH2O and letting drain. 3) Add about 1/4 inch layer of 425-600 µm diameter glass beads (Sigma G9268) to the top of

the resin bed to protect the top surface from disruption as fluids are added. 4) Charge the column by adding 10 ml of 1X Charge buffer and letting drain. The color of the

resin should turn from white to blue. 5) Wash the column by adding 6 ml of 1X Bind buffer and letting drain 6) Affix a closed stopcock to the bottom of the column. Add a few ml of culture supernatant to

the top of the resin, at first by slowly running it down the inside of the column to prevent disruption of the resin. Secure the cap on top of the column.

7) Place the bottle containing the culture supernatant on a shelf a few feet above the top of the column. Set up a length of small diameter flexible tubing connecting the bottle of culture supernatant to the cap on top of the column. We recommend using 1/16 in (inner diameter) tubing (Tygon ALC00002) with a short (~2 in) length of 1/8 in (inner diameter) tubing

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(Tygon ALC00007) at the end to act as a coupling for attachment to the nipple on the end of the column cap. Make sure that the lowest point in the tubing loop hangs below the bottom of the column. This will prevent the column from running dry after the bottle has emptied.

8) To prime the tubing, remove the tubing from the nipple on the column cap. Using a syringe, draw fluid from the bottle to near the end of the tubing, then clamp the tubing about 2 inches from the end with a hemostat. Remove the syringe, then re-attach the end of the tubing to the nipple on the column cap.

9) Unclamp the top tubing and open the stopcock. The fluid should start dripping from the bottom of the column. Use the stopcock to adjust the flow rate to about 2 ml per minute. Let the column drip overnight, collecting the flow-through fraction in a new bottle. The safety loop should prevent the resin from drying out once the supernatant bottle has emptied. Leaving the resin in a dry state is bad.

10) Once the supernatant has passed over the column, close the stopcock, clamp the top tubing and remove the cap. Transfer the column on the ringstand to a bench in the lab. Open the stopcock to let any remaining supernatant drain from the resin.

11) Wash the column with 5 ml 1X Bind buffer (10 mM imidazole). Let the top of the column just go dry.

12) Quickly add 12 ml of 1X Elute buffer (1M imidazole) to the top of the column. Collect all 12 ml. Close the stopcock.

13) Regenerate the column by adding 6 ml 1X Strip buffer and letting drain. The resin should change in color from blue to white. Close the stopcock and add another 2 ml to the top of the column. Replace the cap on the column, and put the column back in the cold room for storage.

Note: At this point the eluted sample contains biotinylated p:MHCII, unbiotinylated p:MHCII, and insect cell proteins that bind to His-Bind for unknown reasons. The next step is designed to isolate the biotinylated p:MHCII away from the other proteins. C. Isolate biotinylated p:MHCII on a monomeric avidin column. 1) Remove the Pierce Monomeric Avidin UltraLink column (~2.5 ml bed volume) from the cold room and let warm to room temperature. Use room temperature buffers for procedure 2) Wash the column with 7.5 ml (three resin-bed volumes) of Biotin Blocking and Elution Buffer (2 mM biotin in PBS) to block any non-reversible biotin binding sites on the column. 3) Remove biotin bound to reversible biotin-binding sites by washing with 12.5 ml (five resin-bed volumes) of Regeneration Buffer (0.1M glycine, pH 2.8). 4) Wash the column with 12.5 ml (five resin-bed volumes) of PBS to re-equilibrate for binding. 5) Apply the protein-containing 12 ml eluate from the His-Bind column (II.B.12). Let the sample drip through the column. Note: Binding is only slightly increased by incubation. 6) Wash column with 25 ml of PBS. 7) To elute the bound biotinylated p:MHCII, add 12 ml of Biotin Blocking/Elution Buffer (2 mM biotin in PBS) to the column and collect 1 ml fractions. Measure the absorbance of each fraction at 280 nm (use PBS to obtain a baseline value) and pool the fractions that contain protein, which will usually be fractions 3-6. 8) Regenerate the column by washing with 12.5 ml Regeneration Buffer (0.1M glycine, pH 2.8). 9) Wash with 20 ml of PBS with 0.01% sodium azide and store the column in the cold room.

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10) Add the pooled fractions from step 7 to an Amicon Ultra-15 30 kD concentrating filter (Millipore) and spin at ~1500xg (3,000 rpm in a Beckman tabletop centrifuge) until the volume has been reduced to less than 0.5 ml. A spin of 15 minutes is usually sufficient for this purpose. 11) Add 4 ml of PBS and spin for 15 minutes at 3,000 rpm until the volume has been reduced to 0.2 ml. 12) Add 4 ml of PBS and spin for 15 minutes at 3,000 rpm until the volume has been reduced to 0.2 ml. 13) Add 4 ml of PBS and spin for 15 minutes at 3,000 rpm until the volume has been reduced to 0.2 ml. 14) Add 4 ml of PBS with 0.01% sodium azide (PBSA) and spin for 15 minutes at 3,000 rpm until the volume has been reduced to 0.2 ml. Note: The purpose of the 4 PBS washes is to remove the biotin used to elute the biotinylated p:MHCII. This is a critical step since free biotin will inhibit the tetramerization reaction. Four washes from 4 ml to 0.2 will produce a 160,000-fold dilution of the biotin used to elute the biotinylated p:MHCII, reducing its concentration from 2 mM to 0.0125 µM. This small amount of residual free biotin will be inconsequential because the final p:MHCII concentration will be at least 15 µM. 15) Measure the OD280 using the Nanodrop device and calculate the biotinylated-pMHC concentration. The extinction coefficient for pMHCII is 0.066 cm-1(µM)-1 (i.e. an OD280 of 0.066 corresponds to a concentration of 1 µM). FYI - the molecular weight of pMHCII is 66 kD. Aim for a concentration of at least 15 µM. VI. Assess pMHC biotinylation Since all of the pMHCII that is eluted by biotin should be biotinylated, the concentration of pMHCII calculated in III.C.15 should equal the concentration of biotin. However, it is still worth confirming this fact by doing an SA shift up test. 1) Set up the following reactions in microfuge tubes in a total of 20 µl of PBSA: Tube pMHC per tube SA-PE per

tube 1 10 µg (150 pmol) None 2 10 µg 37.5 pmol 2) Add 6.7 µl of 4X native PAGE sample buffer (see recipes) to each tube, then run 20 µl of each sample on a 4-15% Tris-glycine acrylamide gel using non-reducing SDS-PAGE running buffer (see recipes). Use 10 µl of Precision Plus Kaliedoscope molecular weight markers (BioRad 161-0375) in the outer lanes. 3) Run at 150V about 1 h. 4) Transfer gel to a small tray and cover gel with Coomassie staining solution (see recipes). Note that the gel is flimsy and easily torn. 5) Microwave for 30 seconds, then place on a shaker for 15 minutes. 6) Pour Coomassie staining solution into stock bottle for re-use.

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7) Add destaining solution (see recipes), swirl the gel, dump out the destaining solution, add fresh destaining solution, microwave for 30 seconds, then place on a shaker with a balled up Kimwipe the corner for about an hour until the pMHCII band becomes visible. 8) Use the Odyssey Infrared Imager to detect the blue bands using the 700 nm setting. The Tube 1 sample should give a single band at 66 kD, corresponding to biotinylated pMHCII (see gel below). No band should be present in the Tube 2 sample because all of the biotinylated pMHCII should be bound to SA-PE and pulled up to a high position in the gel. If there are bands in the Tube 1 sample other than the 66 kD band, then the assumption that all of the protein is biotinylated pMHCII is incorrect. In this case you can do densitometry to determine the amount of protein in Tube 1 that is biotinylated pMHCII and correct the amount of SA-PE that will be needed to saturate the biotinylated pMHCII. For example if only half of the protein in Tube 1 runs at 66 kD, then only 75 pmol is biotinylated pMHCII, and 18.75 pmol of SA-PE will be needed to make tetramer. 9) If there is only one band in Tube 1 and it runs at 66 kD and it almost completely disappears in Tube 2, then it is accurate to assume that all of the pMHCII is biotinylated and the concentration of pMHCII calculated in III.C.15 equals the concentration of biotin. This concentration can then to used to make tetramer. Add 4.5 moles of biotinylated pMHCII to 1 mole of SA-PE to be on the safe side. The tetramer should form instantaneously after mixing the biotinylated pMHCII and SA-PE. Dilute the mixture with PBSA to adjust the PE concentration to 1 µM. The tetramer is now ready for use. Note: A very faint pMHCII-biotin band in the lane with the sample mixed with SA-PE indicates that the SA-PE was saturated a shown in lane 2 below. This is the desired outcome. If the pMHCII-biotin band completely disappears then you cannot be sure that the SA-PE was saturated. In this event, repeat the experiment but add 30, 25, and 20 nmols of the SA-PE to 150 nmol of pMHCII-biotin to find the saturation point to use to make tetramer.

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Recipes Complete S2 Medium: Schneider's Drosophila Medium 500 ml (Invitrogen 11720) Fetal Bovine Serum 10 % 50 ml (heat-inactivated @ 56C 20 min) Pen/Strep 100 U/ml each 5 ml of 100X Gentamycin 20 µg/ml 1 ml of 10 mg/ml Serum Free Medium (SFM): Express Five Serum Free Medium 1 L (Invitrogen 10486-025) Pen/Strep 100 U/ml each 10 ml of 100X Gentamycin 20 µg/ml 2 ml of 10 mg/ml 1M CuSO4: CuSO4 (copper (II) sulfate) pentahydrate 1 M 12.5 g (Sigma C8027) dH2O qs 50 ml pass through sterile 0.2 µm filter 1 mg/ml D-biotin: D-biotin 1 mg/ml 50 mg (Pierce 29129) dH2O 49.8 ml 1N NaOH ~190 µl to pH 7 pass through sterile 0.2 µm filter 4x Native Lam's Sample Buffer: Tris HCl pH 6.8 200 mM 5 ml of 1 M Glycerol 60 % 15 ml Bromophenol Blue 20 µg/ml 500 µg dH2O qs 25 ml 10X Tris-Glycine SDS-PAGE Running Buffer: Tris-base 250 mM 30 g Glycine 1.9 M 144 g SDS 0.035 M 10 g dH2O qs 1.0 L Coomassie Staining Solution

500 ml Methanol 400 ml Ultrapure water 100 ml Glacial Acetic Acid

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2.5 g Coomassie Brilliant Blue R-250 Coomassie Destaining Solution

785 ml Ultrapure water 165 ml Ethanol 50 ml Glacial Acetic Acid