The Streptamer Principle

Strep-tags are short peptides with high binding selectivity for Strep-Tactin, an engineered streptavidin. The binding affinity of e.g. Strep-tag II to Strep-Tactin (Kd = 1 µM) is nearly 100 times higher than to streptavidin. Strep-tags may be fused to recombinant proteins which allows efficient one-step purification of such fusion proteins on immobilized Strep– Tactin under physiological conditions, thus preserving their bioactivity.

As the Strep-tag binds to the biotin binding pocket of Strep-Tactin, purified proteins may be mildly eluted from the column by the addition of minute amounts of biotin.

A special application of the Strep-tag:Strep-Tactin technology is the oligomerization of MHC I-Strep-tag fusion proteins on Strep-Tactin. These complexes may be used for the efficient antigen specific staining of T-cells by using modified Strep-Tactin being labeled by a fluorescent probe or a magnetic particle. After separation of the stained T-cells from non-stained cells by florescence activated cell sorting (FACS) or by a magnetic field, respectively, the staining may be efficiently removed by the addition of biotin.

This removal of the Strep-Tactin backbone leaves monomeric MHC I-Strep-tag fusion proteins on the surface of the T-cell. As the monovalent MHC I:T-cell receptor interaction is weak, MHC I-Strep-tag fusion proteins spontaneously dissociate from the T-cell receptor and may be removed from the T-cells simply by washing. Keeping stained cells at 4 °C together with rapid and complete dissociation of Streptamers from the T-cells after purification assures the isolation of fully functional, non-induced T-cells.

Experimental procedure

Routinely approximately 5×106 cells are stained using 0.75 µg Strep-Tactin-PE (5 µl) and1 µg MHC I (4 µl) in a final volume of 50 µl. All steps, the staining of the cells as well as the following dissociation of Streptamers, have to be performed at 4°C. Please make sure that all your reagents and the cells have reached the temperature before starting the protocol.

Titration

If the staining protocol is not suitable for your application, a titration of the MHC should be performed.

  • Test 0.75 mg Strep-Tactin-PE with 1 mg, 2 mg and 5 mg MHC I, respectively.
  • The assay can be conducted in a 96-well round bottom microplate.
  • All incubations are carried out in the dark to protect PE from light.

Protocol for the staining of T-cells with Streptamers

All steps have to be performed at 4°C

  1. Incubate 0.75 µg (5 µl) Strep-Tactin-PE and 1 µg (4 µl) MHC in a final volume

of 50 µl Buffer IS for 45 minutes.

  1. Add the pre-incubated Strep-Tactin-PE/MHC I preparation to the cell pellet.
  2. Incubate for 45 minutes.
  3. Wash cells twice with 200 µl Buffer IS.
  4. Cells are ready for FACS-analysis or FACS-sorting.

Streptamer Magnetic T-cell Labeling and Isolation via MACS

Purification scheme

In a first step, T-cells are labeled with a magnetic Streptamer complex according to their antigen specificity, then stained T-cells are separated from other cells by a magnetic field and such purified T-cells are eluted and released from the Streptamer complex by the addition of biotin (vitamin H) to yield a functional, non-induced antigen specific T-cell preparation

Recommendations for isolating T-cells using Strep-Tactin magnetic beads and recombinant MHC I proteins fused to Streptag

Experimental procedure

The procedure is optimized to isolate antigen-specific T-cells from 2×107 peripheral blood mononuclear cells (PBMC). Some cells like monocytes or natural killer cells may also be co-purified due to their ability to bind MHC and can be depleted before the actual T-cell isolation.

For human blood:

  • Anticoagulant treatment
  • Ficoll gradient
  • T-cell isolation

For mouse blood:

  • Anticoagulant treatment
  • Ficoll gradient
  • CD8+ enrichment
  • Optional: NK-cell depletion
  • Antigen-specific T-cell isolation

All the steps from isolation of cells as well as the following dissociation of Streptamers . have to be performed at 4°C. Please make sure that all your reagents and the cells have reached the temperature before starting the protocol.

Reagents

  • Streptamer Magnetic Beads:250 µl (suff. for 1X108 cells)
  • Streptamer Magnetic Beads: 1.25 ml (suff. for 5X108 cells)
  • MHC I-Strep-tag: 200 µl (suff. for pur. of 5X108 human or 2.5X108mouse cells)
  • Solution set for magnetic beads includes: for 5 preps (2X107 cells each)
  • Buffer IS
  • Biotin stock solution
  • Nylon filter mesh (100 µm)
  • Blood or T-cell sample
  • Miltenyi columns and separators
  • Centrifuge
  • Test tubes

For optional staining and FACS analysis

  • EMA
  • Strep-Tactin PE and recombinant MHC I proteins fused Strep-tag
  • CD8-PE or CD8-FITC antibody
  • CD3-FITC or CD3-APC antibody
  • FACScan

For optional Ficoll gradient centrifugation

  • Ficoll
  • PBS or balanced salt solution
  • Pasteur pipettes
  • Syringe with needle
  • Silicone solution
  • Distilled water

For optional CD8+ enrichment

  • CD8+ T-cell Isolation Kit

Experimental procedure

The procedure is optimized to isolate antigen-specific T-cells from 2×107 peripheral blood mononuclear cells (PBMC). The chapters describe the isolation of these cells and depletion of non-T-cell populations, respectively.

Anticoagulant treatment

  1. EDTA is added to a final concentration of 20 mM
  2. Same volume of PBS or balanced salt solution is added to the EDTA-blood Other anticoagulants have been used like heparin, citrate, acid citrate dextrose, citrate phosphate dextrose.

Ficoll gradient centrifugation

Procedure for isolation of lymphocytes from blood samples.

  1. The required volume of Ficoll (3 ml for 4 ml diluted anticoagulated blood sample) is aseptically withdrawn using a syringe.
  2. Ficoll-Paque Plus (3 ml) is added to a centrifuge tube
  3. Carefully layer diluted blood sample (4 ml) on Ficoll-Paque Plus. When layering the sample do not mix Ficoll and diluted blood sample.
  4. Centrifuge at 400x g for 30-40 minutes at 18-20°C
  5. Draw off the upper layer using a clean Pasteur pipette, leaving the lymphocyte layer undisturbed at the interface. Care should be taken not to disturb the lymphocyte layer. The upper layer of plasma, which is essentially free of cells, may be saved for later use.
  6. Using a clean pasteur pipette transfer the lymphocyte layer to a clean centrifuge tube. It is critical to remove all of the interface but a minimum amount of Ficoll and supernatant. Removing excess Ficoll causes granulocyte contamination, removing excess supernatant results in platelet and plasma protein contamination.
  7. Add at least 3 volumes of balanced salt solution to the lymphocytes in the test tube.
  8. Suspend the lymphocytes by gently drawing them in and out of a the Pasteur pipette
  9. Centrifuge at 10-100 x g and 18-20°C for 10 minutes.
  10. Remove the supernatant
  11. The lymphocytes should now be suspended in an appropriate medium and can be frozen

Optional: CD8+ enrichment

CD8+ enrichment is recommended for the isolation of mouse antigen specific T-cells only.

Magnetic labeling

  1. Determine cell number
  2. Centrifuge cell suspension at 300 x g for 10 minutes. Pipette off supernatant completely
  3. Resuspend cell pellet in 40 µl of buffer per 107 total cells.
  4. Add 10 µl of biotin antibody cocktail per 107 total cells.
  5. Mix well and incubate for 10 minutes at 4-8°C.
  6. Add 30 µl of buffer per 107 total cells.
  7. Add 20 µl of anti biotin MicroBeads per 107 total cells.
  8. Mix well and incubate for an additional 15 minutes at 4-8°C.
  9. Wash cells with buffer adding 10-20 x labeling volume and centrifuge at 300 x g for 10 minutes. Pipette off supernatant completely.
  10. Resuspend cells in 500 µl buffer. Up to 108 total cells can be resuspended in 500 µl, larger numbers require an accordingly larger volume of buffer.
  11. Proceed to magnetic separation.

Magnetic separation with MS or LS columns

  1. Place column in the magnetic field of a suitable MACS separator.
  2. Prepare column by rinsing with appropriate amount of buffer: MS: 500 µl LS: 3 ml
  3. Apply cell suspension onto the column. Collect flow-through. Allow cells to pass through and collect effluent as fraction with unlabeled cells, representing the enriched CD8+ T-cell fraction.
  4. Wash column with appropriate amount of buffer. To wash the column, buffer is added three times when column reservoir is empty: MS: 500 µl LS: 3 ml. Collect entire effluent and pool with flow-through (step 3). This fraction represents the CD8+ T-cells.
  5. Optional: Elute retained cells outside of the magnetic field. This fraction represents the magnetically labeled non-CD8+ T-cells.

Magnetic separation with the autoMACS separator

  1. Prepare and prime the autoMACS
  2. Place tube containing the magnetically labeled cells in the autoMACS separator and choose program “Deplete”
  3. Collect negative fraction. This fraction represents the enriched CD8+ T-cells.
  4. Optional: Collect positive fraction. This fraction represents the magnetically labeled non-CD8+ T-cells.

Isolation of antigen specific T-cells with Streptamer magnetic beads

Protocol for human cells

When working with anti-coagulated peripheral blood or buffy coat, PBMC should be

isolated by density gradient centrifugation first. This Protocol is adapted for 2 x 107 cells. Higher cell numbers require larger amounts of beads and MHC.

  1. Thaw frozen cells in normal growth medium. Make sure concentration of DMSO is below 1%. Cells grown in medium containing less than 10% FCS should be thawed in medium containing 10% FCS instead of their normal growth medium
  2. Wash cells in buffer IS and resuspend in 10 ml (use 300 g for each centrifugation).
  3. Pass cells through enclosed 100 µm nylon mesh. This is necessary to remove cell clumps which may clog the columns.
  4. Determine cell number, take sample (before separation) and place cells on ice. 5. Incubate 50 µl magnetic beads, 8 µl MHC, and 90 µl buffer IS at least 45 minutes at 4°C (or over night).
  5. Place MiniMACS column in the magnetic field and prepare column by rinsing with 2 ml buffer IS.
  6. Add 1 ml buffer IS to MHC/magnetic beads solution and load on MS column. To wash away unbound MHC, magnetic beads are bound and washed on a MS column.
  7. Wash with 2 ml buffer IS.
  8. Add 250 µl buffer IS and elute retained beads outside of the magnetic field into a fresh vial and firmly flush out the beads using the supplied plunger supplied with the column.
  9. Centrifuge cell suspension (300 g) and resuspend in 250 µl magnetic beads/MHC solution. Incubate 45 minutes on ice.
  10. Add 1.5 ml buffer IS, centrifuge cell and beads mixture and wash carefully once with 2 ml buffer IS. This is necessary to eliminate unbound magnetic beads which may trap cells on the column un-specifically.
  11. Resuspend in 2 ml buffer IS. Proceed to magnetic separation.

Isolation of antigen specific T-cells with Streptamer magnetic beads

Protocol for mouse cells

When working with cells from spleen or lymph node cells, be careful to resuspend cells completely. Other organ preparations may require protease digestion and/or gradient centrifugation. Mouse T-cell separation protocol is established for 2 x 107 cells. Higher cell numbers require larger amounts of beads and MHC.

  1. Centrifuge cells twice 10 minutes at 300 g at 4°C and resuspend in 10 ml buffer IS, respectively.
  2. Pass cells through enclosed 100 µm nylon mesh. This is necessary to remove cell clumps which may clog the columns.
  3. Determine cell number, take sample (before separation, approximately 1-5 x 105 cells are required per staining) and place cells on ice.
  4. Incubate 50 µl magnetic beads and 16 µl MHC and 80 µl buffer IS at least 45 minutes at 4°C (or over night).
  5. Place MiniMACS column in the magnetic field and prepare column by rinsing with 2 ml buffer IS.
  6. Add 1 ml buffer IS to MHC/magnetic beads solution and load on MS column. To wash away unbound MHC, magnetic beads are bound and washed on a MS column.
  7. Wash with 2 ml buffer IS.
  8. Add 250 µl buffer IS and elute retained beads outside of the magnetic field into a fresh vial and firmly flush out the beads using the supplied plunger supplied with the column.
  9. Centrifuge cell suspension and resuspend in 250 µl magnetic beads/MHC solution. Incubate 45 minutes on ice.
  10. Add 1.5 ml buffer IS, centrifuge cell and beads mixture 10 minutes at 300 x g at 4°C and wash once by resuspending in 2 ml buffer IS and centrifuging as above. This is necessary to eliminate unbound magnetic beads which may trap cells on the column un-specifically.
  11. Resuspend in 2 ml buffer IS. Proceed to magnetic separation.

Magnetic separation on LS and MS columns

  1. Place LS column in the magnetic field and prepare column by rinsing with 3 ml buffer IS.
  2. Apply cell suspension onto the column. Allow cells to pass through and collect effluent for later analysis (.Flow-through.).
  3. Wash column with 2 x 3 ml buffer IS.
  4. Add 2 x 3 ml buffer IS and elute retained cells outside of the magnetic field into a fresh vial (optional: take sample for analysis).
  5. Rinse MS column with 0.5 ml and apply cells eluted from LS column.
  6. Wash column with 2 x 2 ml buffer IS.
  7. Add 2 x 3 ml buffer IS and elute retained cells outside of the magnetic field into a fresh vial, take sample for analysis (eluted fraction, should contain the isolated T-cells).

Magnetic separation with the autoMACS separator

  1. Prepare and prime the autoMACS
  2. Place tube containing the magnetically labeled cells in the autoMACS separator and choose program “PosseId”.
  3. Collect positive fraction (outlet port “pos2”). This fraction represents the magnetically labeled antigen specific T-cells.
  4. Optional: Collect negative fraction (outlet port “neg1”). This fraction represents mostly T-cells specific for other antigens and other cell types.

 Dissociation of Streptamers with D-biotin

  1. Centrifuge eluted cells and resuspend in 2 ml buffer IS containing 1 mM Dbiotin and incubate for 20 minutes.
  2. Centrifuge cells and resuspend in 2 ml buffer IS containing 1 mM D-biotin and incubate for another 20 minutes.
  3. Wash cells 4 x with 5 ml buffer IS.

Staining of T-cells with Streptamers

To evaluate the purity of the enriched antigen-specific T-cells, fractions can be analyzed by flow cytometry. Live/dead stain can be analyzed with ethidium monazid

bromide (EMA), CD8+ T-cells can be detected using a CD8-PE antibody, and optionally a T-cell marker like CD3 can be detected with, e.g. CD3-FITC antibody. The antigen specific fraction of these cells can be detected by using MHC in combination with a Strep-Tactin-PE conjugate. When secondary staining of CD3 or CD8 is desired add the respective antibody 25 minutes after the addition of the Strep-Tactin-PE/MHC I complex to the cells so that its incubation will last 20 minutes (total incubation of the Strep-Tactin-PE/MHC I with the cells = 45 minutes).

Buffer Composition

  • Phosphate buffered saline (PBS): 8.06 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, pH 7.4
  • Buffer IS: 0.5 % BSA (w/v), in PBS pH 7.4
  • Biotin stock solution isotonic 100 mM biotin/NaCl pH 7.4