Altman Lab: Class I MHC Tetramer Preparation: Folding Reaction Concentration and Biotinylation

At the end of the incubation of the class I MHC/peptide folding reaction, the folded protein is concentrated by ultrafiltration, the buffer is exchanged on disposable PD-10 columns containing G25 resin, and the BSP tag is biotinylated by the BirA enzyme. In the past, we would perform the biotinylation reaction after purification of the folded protein on a Sephacryl S300 column (or a Superdex S200 column), but we find that the protocol described here allows us to omit a couple of subsequent steps, saving time and probably improving the final yield.

Materials and Equipment

Reagents:

1mg/ml Leupetin stock
1000x Pepstatin
100mM PMSF
100mM ATP (5mM final concentration)
100mM Biotin
BirA enzyme (Avidity, or homegrown)

Biotinylation Buffer:

100mM Tris (pH 7.5)
200mM NaCl
5mM MgCl2
Adjust pH to 7.5
Note that Avidity recommends a bicine-based BirA reaction buffer at pH 8.3. Avidity's buffer was derived from kinetic optimization experiments with either small molecule substrates, or BSP tagged proteins. We have not switched to the pH 8.3 buffer for several reasons: (1) our buffer works; and (2) we are concerned about the stability of the MHC peptide complexes at pH 8.3, including susceptibility to competing proteolytic reactions. We have not systematically investigated any of this.

Equipment and Supplies

Refrigerated benchtop centrifuge, such as the Beckman xxxx.
High speed centrifuge, with a rotor that can take 250 ml bottles, such as the Beckman xxxx.
Nitrogen gas cannister and appropriate regulator
Millipore Amicon 8400 Stirred Cell (Cat. No. 5124)
Biomax PB Polyethersulfone Ultrafiltration Membranes 10,000 NMWL (Cat. No. PBGC07610), Millipore
Amicon Ultra-15 PLGC Centrifugal Filter Unit 10,000 NMWL (Cat. No. UFC901024, pack of 24)
PD-10 desalting columns from GE/Amersham/Pharmacia (whoever they are this week!) (cat# 17-0851-01)
LabMate PD-10 Buffer Reservoir (cat# 18-3216-03)

Concentration Protocol

Centrifuge folding reaction to remove precipitated protein.
Prior to concentration, separate the folding reaction into three equal parts and distribute among three centrifuge tubes. Centrifuge the folding reaction at 13000 rpm for 15min. Use Beckman 250ml tubes with appropriate Beckman lids.
This step removes precipitated protein that can clog the ultrafiltration membrane in the next step.
Assemble Amicon 8400 concentrator.
Place a Biomax membrane (NMWL: 10k) in a clean Amicon 400ml stir cell (model 8400) with the shiny side of the membrane facing up. Place an O-ring gasket between the base of the cell and the membrane to secure the membrane in place. Screw the body of the cell onto the base with the volume scale on the same side as the eluate tubing protuberance.
Add folding reaction supernatant to concentrator.
Add the centrifuged supernatant of the folding reaction to the concentrator cell and place the lid of the cell onto the body of the concentrator. Confirm that the gasket in the lid is both present and intact.
Attach concentrator to N₂ gas tank.
Attach the lead from the N₂ gas tank to the lid of the concentrator. Place the pressure-release switch in the vertical position. (This switch can be turned horizontally to release the pressure in the concentrator.) Turn on the gas slowly such that the pressure increases to a maximum of 60 to 65psi.
Concentrate the folding reaction to 7.5ml and stop the N₂.
Note: To concentrate the folding reaction to precisely 7.5ml, measure the volume of the concentrate after it has fallen well below the 50ml mark on the concentration flask (circa 20ml). Use a small graduated vessel (i.e. 15ml polypropylene falcon tube) in lieu of the collecting bottle and monitor the progress of elution.

Buffer Exchange Protocol

At this point, the folding buffer is rapidly exchanged, and is replaced with BirA reaction buffer.

Centrifuge concentrated folding reaction.
Place the concentrate of the folding reaction into a 25ml centrifuge tube. Centrifuge the concentrate for 10min at 15300 rpm at 4°C.

This will remove any precipitate that might have accumulated during the concentration of the folding reaction. Often, we will perform this step by aliquotting the entire sample to several microcentrifuge tubes, followed by pooling of the supernatant.
Prepare three PD-10 Sephadex G-25 M columns.
Equilibrate each of the three PD-10 (Amersham Pharmacia #17-0851-01) columns by adding 5ml of biotinylation buffer to each tube five times (25ml total volume per column).

Note that Amersham/Pharmacia now sells convenient 25 ml reservoirs for equilibration of their PD-10 columns.
Add 2.5ml of protein to each column.
Allow the flow-through to go into the waste.
Place a 15ml polypropylene tube below one of the three columns
This tube will catch the flow-through of each of the tubes.
Successively, add 3.5ml biotinylation buffer to each column.
The biotinylation buffer will elute the protein from the column. Prior to each successive addition of biotinylation buffer, place the polypropylene tube below the column. Alternatively, elute the protein into three tubes and pool the contents. The protein should be at a final total volume of 10.5ml.

Biotinylation

Add the following to the eluate (10.5ml):

500 µl ATP (100mM stock)
40 µl Biotin (100mM stock)
10 µl Leupeptin (1000x stock)
10 µl Pepstatin (100x stock)
20 µl PMSF (0.1M stock)
20 µl BirA enzyme (stock at ~ 1 mg/ml).
We recognize that with this protocol, we do not know the enzyme/substrate ratio. However, we still believe that biotinylation at this stage is convenient and saves time in the end.

Mix by gentle inversion and incubate at room temperature for 12 hours (overnight).
If the temperature of your lab is variable (as is ours!), you might consider placing your sample in a water bath maintained at 20 °C in a cold room.

A slight precipitate might form during the BirA reaction. This is not cause for alarm. The precipitate is removed by centrifugation at the beginning of the next sub-protocol.