Tetramer Protocols: The Folding Reaction

This page contains instructions for setting up the in vitro folding of class I MHC/peptide complexes. The protocol, used in the original preparation of MHC tetramers from Altman et al., was derived from Garboczi and Wiley.

Reagents & Solutions

Reagents for 500ml reaction:

Folding buffer (pH 8.3):

Injection Buffer (pH 4.2):

Day 1

  1. Chill folding buffer.

    In a 1L glass Erlenmeyer flask equipped with a large stir-bar, chill 500ml of folding buffer to 10°C.

  2. Add first three reagents to the folding buffer.

    Add the reduced glutathione, oxidized glutathione and PMSF to the cold folding buffer.

  3. Dissolve the peptide in DMSO.

    Weigh 15mg of peptide and add it to 500ml DMSO in an Eppendorf tube.

    Note: If the peptide is insoluble in DMSO, add successive small amounts of TCA (100% solution) (~.05ml) until it dissolves. Alternatively, it is possible to use TFA.

  4. Add the peptide to the stirring reaction.
  5. Load the inclusion bodies into two syringes.

    Load 500 nmol heavy chain and 1000 nmol hub2m into two separate disposable 3cc syringe using a 20gauge needle.

  6. Place the folding reactions on a stir plate and inject the inclusion bodies.

    Place the folding reaction on a stir plate set at high speed. Replace the 20g needle with a 26g needle. Forcefully inject the heavy chain and the light chain into the reaction as close to the stirring bar as possible.

  7. Incubate the folding reaction overnight at 10°C.

    Shake the folding reaction in a refrigerated incubator/shaker at 70rpm and 10°C and incubate for 1-3 days.

    If you do not have access to equipment that can maintain the temperature at 10 °C, you can incubate the folding reactions at 4 °C, such as on a magnetic stir plate in a cold room.

Day 2

  1. In the morning, load and inject heavy chain into the folding reaction. Load 500nmol heavy chain into a 3cc syringe using a 20gauge needle.

    Inject the heavy chain forcefully into the reaction as close to the stirring bar as possible.

  2. Store the folding reaction at 70rpm and 10°C all day.
  3. In the evening, load and inject heavy chain into the folding reaction.

    In the evening, load 500nmol heavy chain into a 3cc syringe using a 20g needle. Inject the heavy chain into the reaction as described above.

  4. Incubate the folding reaction overnight at 10°C.

    Shake the folding reaction at 70rpm and 10°C. Store overnight.

    It is possible to incubate the folding reactions for a few additional days; deleterious side reactions—such as proteolysis of the BSP tag—appear to be very slow in folding buffer.