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. 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.
Preparation of Inclusion Bodies
Refolding of KLRG1
Concentration of Refolding Reactions
Purification of KLRG1 tetramer
Representative Results
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.
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.
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.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
BirA Enzyme and Buffer | Biotinylation Kit | Avidity | BIRA500 | |
Complete Mini Protease Inhibitor Tablets, EDTA Free | Reagent | Roche | 11 836 170 001 | or equivalent |
Amicon Stirred Cell | Equipment | Millipore | Model #8400 | or equivalent |
YM10 NMWL 10K ultrafiltration membrane | Filtration Supply | Millipore | 13642 | |
15 ml YM10 concentrator | Filtration Supply | 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 |