Name: A Protocol for the Production of KLRG1 Tetramer (Video) | JoVE Journal
Uploaded: 2010-01-12T16:01:00.000+00:00
Duration: 7 min 24 s
Description: Learn to produce KLRG1 tetramers for analyzing ligand-receptor interactions in immunology research. This bacterial expression protocol enables detailed study of immune regulation mechanisms.

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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.

Preparation of Inclusion Bodies
<ol>
 <li>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. </li>
 <li>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.</li>
 <li>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.</li>
 <li>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).</li>
 <li>Sonicate the solution on ice for 1.5 min and centrifuge the lysates at 5,000 RPM for 10 min at 4°C.
 <ol class="lalpha">
 <li>Decant the supernatant.</li>
 <li>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).</li>
 <li>On ice, sonicate solution for 1.5 min until the pellet is completely resuspended.</li>
 <li>Centrifuge the samples at 5,000 RPM for 10 min at 4°C.</li>
 <li>Repeat the wash 5 times.</li>
 </ol>
 </li>
 <li>Repeat 5b with wash buffer without Triton X-100, centrifuge and resuspend in 4 ml of TE Buffer.</li>
 <li>Take the wet weight inclusion bodies slurry and store in TE buffer at a concentration of 30 to 100 mg/ml.</li>
</ol>
Refolding of KLRG1
<ol>
 <li>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. </li>
 <li>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.
 <ol class="lalpha">
 <li>Melt the volume of 50 mg of inclusion bodies slurry in 7 M GnHCl with 10 mM β-mercaptoethanol.</li>
 <li>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).</li>
 <li>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. </li>
 </ol>
 </li>
 <li>Adjust volume with injection buffer (3 M GnHCl, 10 mM NaAcetate, pH 4.2,10 mM EDTA).</li>
 <li>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.</li>
 <li>Continue overnight stirring at low speed at 4°C. </li>
</ol>
Concentration of Refolding Reactions
<ol>
 <li>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. </li>
 <li>Concentrate to ≤2 ml using a Centriplus YM-10 10K MWCO (Millipore). </li>
</ol>
Purification of KLRG1 tetramer 
<ol>
 <li>Setup the AKTAFPLC at 4°C according to GE Healthcare manuals.</li>
 <li>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).</li>
 <li>Concentrate to 1 ml using Centriplus YM-10 10K MWCO (Millipore).</li>
 <li>Use BirA enzyme according to Avidity s protocol to biotinylate the KLRG1 monomer.</li>
 <li>The free biotin is eliminated by size exclusion chromatography as in 2.</li>
 <li>KLRG1 molecule is tetramerized by mixing KLRG1 monomer with 4 fold molar excess PE conjugated streptavidin (BD Biosciences).</li>
</ol>
Representative Results
<a href="https://www.jove.com/files/ftp_upload/1702/1702fig1.jpg"><img src="/files/ftp_upload/1702/1702fig1tmb.jpg" alt="Chromatography graph; compound separation peaks; analytical chemistry; retention times labeled." border="0" data-altupdatedat="1788235205000" data-alt="Figure 1"></a> 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.

<ol><li>Tessmer, M. S., Fugere, C., Stevenaert, F., Naidenko, O. V., Chong, H. J., Leclercq, G., Brossay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((KLRG1+binds+cadherins+and+preferentially+associates+with+SHIP-1%5BTitle%5D)+AND+%22Int+Immunol%22%5BJournal%5D)">KLRG1 binds cadherins and preferentially associates with SHIP-1.</a> Int Immunol. 19, 391-400 (2007).</li>
<li>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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cutting+edge%3A+identification+of+E-cadherin+as+a+ligand+for+the+murine+killer+cell+lectin-like+receptor+G1%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1.</a> J Immunol. 176, 1311-1315 (2006).</li>
<li>Ito, M., Maruyama, T., Saito, N., Koganei, S., Yamamoto, K., Matsumoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Killer+cell+lectin-like+receptor+G1+binds+three+members+of+the+classical+cadherin+family+to+inhibit+NK+cell+cytotoxicity%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity.</a> J Exp Med. 203, 289-295 (2006).</li>
<li>Li, Y., Hofmann, M., Wang, Q., Teng, L., Chlewicki, L. K., Pircher, H., Mariuzza, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+natural+killer+cell+receptor+KLRG1+bound+to+E-cadherin+reveals+basis+for+MHC-independent+missing+self+recognition%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.</a> Immunity. 31, 35-46 (2009).</li>
<li>Nakamura, S., Kuroki, K., Ohki, I., Sasaki, K., Kajikawa, M., Maruyama, T., Ito, M., Kameda, Y., Ikura, M., Yamamoto, K., Matsumoto, N., Maenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((K.+Molecular+basis+for+E-cadherin+recognition+by+killer+cell+lectin-like+receptor+G1+%28KLRG1%29%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">K. Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1).</a> J Biol Chem. , Forthcoming Forthcoming.</li>
</ol>

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.

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

a protocol for the production of klrg1 tetramer

A Protocol for the Production of KLRG1 Tetramer

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.

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

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

Hi, I'm Stephanie Resi from the laboratory of Lauren Parce in the Department of Molecular Microbiology and Immunology at Brown University. Today we will show you a procedure for the generation of K LRG one Tetramers. We use this procedure in our laboratory to study K LRG one coherent interactions.Let's get started. First, inoculate four 500 milliliter flasks of TB buffer containing appropriate antibiotics with bacteria expressing the K LRG one plasmid construct and grow at 37 degrees Celsius with shaking when the OD reaches 0.6, angstroms at 600 nanometers induce expression via the T seven promoter with the addition of 0.4 molar IPTG and incubate the culture for an additional four hours shaking at 37 degrees Celsius. Following the four hour incubation, transfer the bacteria to centrifuge tubes and spin the cultures down for 20 minutes at 3000 RPM and four degrees Celsius.If necessary, pellets can be frozen at minus 80 degrees Celsius at this point for use at a later time. Resus suspend the harvested cells in 30 milliliters of Resus suspension buffer Next pool. Bacterial Resus suspensions up to a 60 milliliter volume in a polypropylene beaker.If necessary, bring the total volume up to 60 milliliters with extra te buffer PH eight and stir the mixture at half speed on a hot plate stir while the pooled Resus suspension is stirring at the following solutions. Dropwise to the mixture Lysozyme magnesium chloride D NS one TRITTON X 100. And finally DTT.Transfer the solution to ultracentrifuge tubes using a fisure dismember with ultrasonic converter at 30%amplitude. Sonicate the solution on ice for 1.5 minutes and centrifuge the lysate at 5, 000 RPMs for 10 minutes at four degrees Celsius, decant the supernatant and add 15 milliliters of wash buffer. Repeat sonication as before until the pellet is completely resuspended and repeat centrifugation, repeat the washing fornicating and pelleting steps five times.Finally, resus suspend the pellet using wash buffer that does not contain Triton X 100. Spin down once more and Resus suspend in four milliliters of TE buffer. These are your inclusion bodies.Quantify your inclusion bodies by wet weight. The inclusion body slurry can be kept at four degrees Celsius in te buffer stocks of 30 to 100 milligram per milliliter concentrations. With the inclusion bodies ready, you can now prepare for refolding.Begin the refolding procedure by preparing a fresh one liter bottle of Refolding buffer and stir at low speed at four degree Celsius to chill. Next, you'll prepare seven injections of the inclusion bodies for injection into the refolding buffer. First, melt the stock of inclusion body slurry in a micro centrifuge tube containing a final volume of 1.4 milliliters in seven molar guine hydrochloride plus 10 millimolar beam mercaptoethanol in a water bath at 37 degrees Celsius for 30 to 40 minutes.Vortexing every five minutes. You will know the melting is complete. When the slurry becomes clear.Centrifuge the melted mixture at max speed for 30 minutes in a micro centrifuge at four degrees Celsius. Then being careful not to collect any of the small blackish pellet. Transfer the supernatant to a 15 milliliter centrifuge tube.Adjust the volume to seven milliliters with injection buffer to inject. First, increase the stirring speed of the folding mixture to high. Then add one milliliter of diluted inclusion bodies.Drop by drop with two seconds between each drop only. Add one milliliter of diluted inclusion bodies at a time with an hour in between each injection in between injections. Return stir speed to low o.Continue stirring at low speed at four degrees Celsius overnight. The proteins will then be ready for concentration and purification. To begin concentrating the refolded KLRG one tetramers.First pre-filter the reaction using a point 22 micrometer filter. Then concentrate the reaction from one liter to about 10 milliliters using a millipore Amon stirred cell and a 10 kilodalton nominal molecular weight limit ultra filtration membrane according to manufacturers and S instructions. Then using a Millipore 10 kilodalton molecular weight cutoff ultra filtration membrane concentrate further to less than or equal to two milliliters.Have your chromatography system set up according to manufacturer's instructions. We use the GE healthcare ACTA fast protein liquid chromatography system at four degrees Celsius using a high resolution column. Purify the monomer form of K lrg, one in 20 millimolar heaps and 150 millimolar sodium chloride by size exclusion chromatography.The volume injected should be less than 2%of the total gel volume of the column. Again, using a Millipore 10 kilodalton molecular weight cutoff ultra filtration membrane concentrate further to a final volume of one milliliter ate the KLG one monomer according to manufacturer's instructions. Eliminate the free biotin by purifying as before using size exclusion chromatography to achieve tetramer.Mix the KL LRG one monomers with a fourfold molar excess of FICO rine conjugated streptavidin tetramers are now available for labeling receptor ligands. This graph shows a positive result of separation of Refolded K lrg one proteins using size exclusion chromatography from left to right. The peaks illustrate the multimer dimer and monomer forms of K LRG one.The peak on the far right represents the buffer exchange. Though refolding conditions have to be tested empirically for each protein, other C type lectin receptors should be able to be tetramer using a similar protocol, thus enabling the potential identification of other ligands. We've just shown you how to generate the KLRG one teer when doing this procedure.It's important to remember to check the induction levels and to strictly adhere to the refolding conditions. So that's it. Thanks for watching and good luck with your experiments.

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Learn to produce KLRG1 tetramers for analyzing ligand-receptor interactions in immunology research. This bacterial expression protocol enables detailed study of immune regulation mechanisms.

A Protocol for the Production of KLRG1 Tetramer

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