Tetramer Production: Purification of Biotinylated MHC Protein by S300 Column Chromatography

The steps described on this page and on the MonoQ and biotinylated monomer storage pages should be performed as rapidly as possible, to minimize the potential for proteolysis of the purified, biotinylated MHC protein.

After enzymatic biotinylation, a preparative gel filtration column is run that serves two purposes:

Removal of excess free biotin that can interfere with subsequent tetramer formation; ATP, which can interfere with accurate protein concentration determinations by spectrophotometry, is also removed
Removal of aggregated protein that did not precipitate during previous steps.

We originally used a Superdex 200 26/60 column for this step. A number of years ago, we replaced this column with a Sephacryl S300 26/60 column, which gave us equivalent separation for our particular sample, but cost between 1/5 and 1/10 the price of the Superdex column.

The instructions below are specific to an old Pharmacia FPLC system. Of course, if you have a different chromatgraphy system in your lab, you should adjust the protocol accordingly.

Finally, one comment on the importance of fresh buffers. In our experience, the most common problem with tetramer preps is proteolytic cleavage of the BSP (BirA substrate peptide) by contaminating proteases. Whenever we see this in the lab, the first things that we replace are the biotinyaltion buffer and the column chromatography buffers. Almost always, this solves the problem. Now, we routinely replace the column chromatography buffers at least every two weeks, if not more often.

Reagents

Buffer A: 20 mM Tris, pH 8.0
Buffer B: 20 mM Tris, 500 mM NaCl, pH 8.0
Sephacryl S300 26/60 column (cat# 17-1196-01)

I. Preparation of the FPLC system

Check the levels of buffer A (20mM Tris, pH 8) and buffer B (20mM Tris, pH 8, + 500mM NaCl) in their reservoirs (usually 1 or 2 liter bottles). You should have at least 500 ml of each before proceeding. See notes above about the freshness of the buffers.
Attach the 10ml super loop (brown coil) to valve 1 (positions 2 and 6).
The Pharmacia system comes with a 10 ml piston-style "Superloop". We have replaced this with a 10 ml loop of PEEK tubing that we obtained from Upchurch. We had to replace the fittings with Pharmacia-compatible M6 fittings, which we also obtained from Upchurch. Although this loop takes slightly more manual pressure to load than does the Pharmacia Superloop, it is much easier to rinse in situ, so we don't have to take it off to clean it.

It is essential to take two precautions prior to injecting your protein into these loops. First, you must make sure that it does not contain protein and salt from the previous run that might contaminate your samples. Second, you must make certain that the loop does not contain air bubbles prior to injecting your sample. See the steps below, which describe this thoroughly.

Finally, remember to wash out the sample needle prior to loading your syringe. Always use common sense measures to avoid cross-contamination.
Prior to injecting the sample into the column, centrifuge it for 10 minutes at 15300 rpm and 4 °C.
We usually split the sample into 5-6 separate eppendorf tubes for this, which has the advantage that they are disposable and always handy. This can also be done in one large tube that is rated for this g-force; most Falcon tubes (15 and 50 ml) are not.
p>Carefully transfer pool the supernatants from each tube into a single 50 ml polypropylene tube.

Note: When working at the bench, keep the sample and all buffers/reagents on ice (~4 °C).

II. Washing the loop

Turn keypad on.
Press [MANUAL] button on the FPLC LCC-500 to turn the keypad on.
Set the injection and column selector valve positions.
Press [STEP FORWARD] button three times to open the valve mode. Close valve 1 by setting it to position 2 by entering [1.2] followed by [STORE].

Set the column selector valves to bypass all columns (position 1 on our Valves 4 and 5).
Remove the needle from the injection port.
The needle should only be removed from the injection port when valve 1 is closed. Closing valve 1 prevents air from entering the line and subsequently being injected into the column.
Fill the syringe with wash buffer and insert the needle into the FPLC injection port.
Wash a 10ml syringe with 20mM Tris (pH 8) thoroughly. Fill the syringe with 10ml of 20mM Tris (pH 8), remove all excess air bubbles and insert the needle into the injection port. Do not inject the wash buffer yet!
Open valve 1 and inject the washing reagent.
Open valve 1 by setting it to position 1 (press [1.1] then [STORE]). Inject the 20mM Tris into the 10ml loop being careful not to bend or break the needle.
Set the flow mode.
Press the [STEP BACKWARD] button three times to the flow mode (ML/MIN) and set the flow by pressing [4.0] then [STORE].
Close valve 1.
Press the [STEP FORWARD] button to the valve mode and close valve 1 by setting it to position 2 (as described above).

The loop should drip at a rate of 4ml/min.
Repeat wash.
After the loop has been washed (~5 min.), remove the syringe and repeat the above procedure for washing the loop.

III. S300 Column Chromatography

Close valve 1, fill the needle and insert it into the injection port.
Press [STEP FORWARD] button three times to open the valve mode. Close valve 1 by entering [1.2] followed by [STORE].

Remove the syringe from the injection port and fill it with the biotinylated sample (10.5ml).
Open valve 1 and inject the sample.
Insert the syringe into the injection port and set valve 1 to position 1. Inject the sample into the super loop and press [END]
Set up fraction collector rack.
Fill the large fraction collector rack with 16 disposable 17x100 polypropylene tubes. Place the rack on the fraction collector. Align the arm such that the middle of the arrow is against the center of the first tube.
Set the fraction collector.
Set the fraction size by pressing [FRACTION SIZE], [4] and [STORE RETURN].

Set the peak fraction size by pressing [PEAK FRAC. SIZE], [4] and [STORE RETURN].

Set the delay by pressing[DELAY], [0.17] and [STORE RETURN].
Start the S300 program.
Open the FPLC computer program to run the S300 column. From the main menu, the program is located in the "S300" folder and entitled "S300prep."
If you have more than 10.5 ml of sample, you can let the program run for less than 10 minutes, pause the program on the LCC500 control panel, temporarily set valve 1 to position 1, inject the remaining sample (less than a few ml - the total should be less than 15 ml), return the valve to position 1, and resume the program.
Folded MHC proteins usually elute with a peak at about 200 ml. You should pool the appropriate fractions in a 50 ml Falcon polypropylene tube. Usually, this will be about 4 fractions.

IV. Tris buffer exchange

This step is required to reduce the salt concentration in preparation for chromatography on the MonoQ column.

Add the entire sample to an Amicon Ultra-15 centrifuge filtering device (MWCO: 10k) and centrifuge at 3080rpm at 4 °C until the sample volume reaches approximately 500 µl. Discard the filtrate collected in the bottom half of the device (though you can save it in a 50 ml tube, if you are nervous).
DO NOT DISCARD THE PROTEIN IN THE TOP HALF OF THE DEVICE.
Fill the Ultra-15 centrifuge filtering device with 20mM Tris (pH 8.0), carefully cap tightly, mix by inversion and centrifuge at 3080rpm at 4 °C until the sample volume reaches approximately 500 µl. Again, discard the filtrate in the bottom half of the device.
Repeat the above-described buffer exchange once.
With a glass pasteur pipette, carefully transfer the concentrated sample to an eppendorf tube and centrifuge at 15300 rpm for 15 minutes at 4 °C.
As quickly as possible, proceed to the final “polishing” purification of the MHC protein on the MonoQ. Keep the protein on ice until it is injected on the MonoQ column.

Acknowledgements

Protocol development: The Altman Lab.

Protocol writeup: based extensively upon a version produced by Sophia Albott.