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 JoVE Biology

A Protocol for the Production of KLRG1 Tetramer

1, 1, 1

1Department of Molecular Microbiology and Immunology, Brown University

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    Summary

    This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

    Date Published: 1/12/2010, Issue 35; doi: 10.3791/1701

    Cite this Article

    Terrizzi, S. C., Banh, C., Brossay, L. A Protocol for the Production of KLRG1 Tetramer. J. Vis. Exp. (35), e1701, doi:10.3791/1701 (2010).

    Abstract

    Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.

    Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton.

    Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

    Protocol

    Preparation of Inclusion Bodies

    1. Inoculate 4 x 500 ml flasks of TB each containing 50 μg/ml of carbenicillin and chloramphenicol with an overnight culture of bacteria expressing the KLRG1 plasmid construct. When the OD reaches 0.6 Å at 600 nm, induce with the addition of 0.4 M IPTG and incubate the culture for an additional 4 hours shaking at 37°C.
    2. Resuspend the harvested cells in resuspension buffer pH=8.0 (50 mM Tris-HCl, 25% (W/V) sucrose, 1 mM EDTA, 10 mM DTT). Note: pellets can be frozen at this stage.
    3. Pool no more than 60 ml of bacterial resuspensions in a polypropylene beaker If necessary adjust to 60 ml with TE buffer pH 8.0 and stir mixture at half speed.
    4. To stirring mixture add drop wise: lysozyme (final=1 mg/ml), MgCl2 (final=5 mM), 2 mg DNAse I in 50% glycerol contained 75 mM NaCl, Triton-X100 (final=1%), DTT (final=10 mM).
    5. Sonicate the solution on ice for 1.5 min and centrifuge the lysates at 5,000 RPM for 10 min at 4°C.
      1. Decant the supernatant.
      2. Add 15 ml of wash buffer pH=8.0 (50 mM Tris-HCl, 0.5% Triton X-100, 100 mM NaCl, 1 mM EDTA, 1 mM DTT).
      3. On ice, sonicate solution for 1.5 min until the pellet is completely resuspended.
      4. Centrifuge the samples at 5,000 RPM for 10 min at 4°C.
      5. Repeat the wash 5 times.
    6. Repeat 5b with wash buffer without Triton X-100, centrifuge and resuspend in 4 ml of TE Buffer.
    7. Take the wet weight inclusion bodies slurry and store in TE buffer at a concentration of 30 to 100 mg/ml.

    Refolding of KLRG1

    1. Make 1 L of refolding buffer pH=8.0 (0.4 M Arginine-HCl, 0.1 M Tris pH 8-8.3, 2 mM EDTA, 0.2 mM PMSF, 3 EDTA-free protease inhibitor tablets, 5 mM GSH and 0.5 mM GSSG) and chill to 4°C while stirring.
    2. Prepare the inclusion bodies for injection into the folding mixture: It is ideal to make 5 10 injections each with 0.25 0.5 μM concentration so that the final concentration of the protein will be 2 3 μM.
      1. Melt the volume of 50 mg of inclusion bodies slurry in 7 M GnHCl with 10 mM β-mercaptoethanol.
      2. Keep the inclusion bodies/GnHCl mixture at 37°C for 30 - 40 min and vortex every 5 min to ensure complete melting (slurry will become clear when completely melted).
      3. Centrifuge at max speed for 30 min in microcentrifuge at 4°C and transfer the supernatant to a 15 ml centrifuge tube. Do not to transfer any of the small blackish pellet.
    3. Adjust volume with injection buffer (3 M GnHCl, 10 mM NaAcetate, pH 4.2,10 mM EDTA).
    4. Inject 1 ml of diluted inclusion bodies every hour. When injecting, stir at high speed, add 1 ml of diluted inclusion bodies drop by drop with 2 seconds between each drop. When injection is done, stir at low speed.
    5. Continue overnight stirring at low speed at 4°C.

    Concentration of Refolding Reactions

    1. Following Millipore s protocol, concentrate the 1 L of pre-filtered (0.22 μm filtered) refolding reaction using an Amicon Stirred Cell and YM10 NMWL 10K ultrafiltration membrane (Millipore) to ~10 ml.
    2. Concentrate to ≤2 ml using a Centriplus YM-10 10K MWCO (Millipore).

    Purification of KLRG1 tetramer

    1. Setup the AKTAFPLC at 4°C according to GE Healthcare manuals.
    2. Purify the monomer form of KLRG1 by size exclusion chromatography using a Sephacryl S-200 16/60 high-resolution column (GE Healthcare) in 20 mM HEPES and 150 mM NaCl. (See Figure 1).
    3. Concentrate to 1 ml using Centriplus YM-10 10K MWCO (Millipore).
    4. Use BirA enzyme according to Avidity s protocol to biotinylate the KLRG1 monomer.
    5. The free biotin is eliminated by size exclusion chromatography as in 2.
    6. KLRG1 molecule is tetramerized by mixing KLRG1 monomer with 4 fold molar excess PE conjugated streptavidin (BD Biosciences).

    Representative Results

    Figure 1
    Figure 1. Representative positive results from the AKTA FPLC size exclusion chromatography of the refolded KLRG1. The graph shows the separation of the monomer (83.52 ml), dimer (56.36 ml), and multimer (36.26 ml) form of the refolded KLRG1. The peak at 94.25 ml represents the buffer exchange.

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    Discussion

    In this protocol, it is shown how to purify, biotinylate, and tetramerize KLRG1. KLRG1 tetramer can be used to label KLRG1 ligands by flow cytometry. Although refolding conditions will have to be tested empirically for each protein, other C-type lectins could be tetramerized using a similar protocol. Using tetramerized proteins as probes, ligands to orphan C-type lectin receptors could potentially be identified.

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    Disclosures

    Acknowledgements

    We thank Dr. Naidenko for help in optimizing the refolding conditions. This work was supported by NIH research grant AI58181 to Laurent Brossay. Cindy Banh is supported by NIH NRSA F31 AI080230.

    Stephanie Terrizzi and Cindy Banh contribute equally to this work.

    Materials

    Name Type Company Catalog Number Comments
    BirA Enzyme and Buffer Biotinylation Kit Avidity BIRA500
    Complete Mini Protease Inhibitor Tablets, EDTA Free Reagent Roche Group 11 836 170 001 or equivalent
    Amicon Stirred Cell Equipment EMD Millipore Model #8400 or equivalent
    YM10 NMWL 10K ultrafiltration membrane Filtration Supply EMD Millipore 13642
    15 ml YM10 concentrator Filtration Supply EMD Millipore 4411
    AKTAFPLC Equipment GE Healthcare 18-1900-26
    Sephacryl S-200 16/60 high-resolution column Equipment GE Healthcare 17-1166-01
    Strepavidin PE Reagent BD Biosciences 554061 or equivalent

    References

    1. Tessmer, M. S., Fugere, C., Stevenaert, F., Naidenko, O. V., Chong, H. J., Leclercq, G., Brossay, L. KLRG1 binds cadherins and preferentially associates with SHIP-1. Int Immunol. 19, 391-400 (2007).
    2. Grundemann, C., Bauer, M., Schweier, O., von Oppen, N., Lassing, U., Saudan, P., Becker, K. F., Karp, K., Hanke, T., Bachmann, M. F., Pircher, H. Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1. J Immunol. 176, 1311-1315 (2006).
    3. Ito, M., Maruyama, T., Saito, N., Koganei, S., Yamamoto, K., Matsumoto, N. Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity. J Exp Med. 203, 289-295 (2006).
    4. Li, Y., Hofmann, M., Wang, Q., Teng, L., Chlewicki, L. K., Pircher, H., Mariuzza, R. A. Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition. Immunity. 31, 35-46 (2009).
    5. Nakamura, S., Kuroki, K., Ohki, I., Sasaki, K., Kajikawa, M., Maruyama, T., Ito, M., Kameda, Y., Ikura, M., Yamamoto, K., Matsumoto, N., Maenaka, K. K. Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1). J Biol Chem. Forthcoming Forthcoming.

    Comments

    3 Comments

    Hello, I'm Kyungeun Kim, postdoctoral fellow at MD Anderson. Your protocol is very helpful to me. By the way.. Is this tetramer useful for blocking a interaction between KLRG1-expressing cell and their ligand-expressing cells to reduce KLRG1 signaling? Thank you.
    Reply

    Posted by: AnonymousApril 14, 2010, 12:18 PM

    Hi Kyungeun, thanks for the note, we did some experiments like that but it was not conclusive
    Reply

    Posted by: Laurent B.April 14, 2010, 1:57 PM

    Hello, I am the new guy to protein purification and refolding. Your visualized protocol help me a lot in setting up my own experiment. I have a small question, what is the purpose of the Inject buffer when dilute inclusion bodies into refolding buffer. I also notice that some other protocols (as www.microbiology.emory.edu/altman/jdaWebSite_v3/p_tetFolding.shtml) have a slight different injection buffer recipe in the concentration of GnHCl(3mM). And another question, Did the amount of inject buffer add into inclusion bodies/GnHCl mixture affect the refolding results? Thanks Lu Yi from China
    Reply

    Posted by: AnonymousNovember 12, 2010, 3:28 AM

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