3 3 cholamidopropyl dimethylammonio 1 propane sulfonate

Restriction endonucleases and DNA-modifying enzymes were purchased from New England BioLabs, Inc. (Ipswich, MA). Soybean phosphatidylcholine was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate and other reagents were purchased from Sigma (St Louis, MO).

For tetramers, 20 μg CD1b monomers obtained from the National Institute Health (NIH) tetramer facility were loaded with 32 μg PG (Avanti lipids), GMM (Bill and Melinda Gates Foundation Lipid Bank) or synthetic BbGL-II (a gift from J. Kubler-Kielb, NIH). Lipids were dissolved in citrate buffer pH 4.5 with 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Sigma) and incubated overnight at 37°C. After incubation the pH was neutralized by adding Tris pH 8.5. As negative control CD1b monomers were treated in the same way without adding exogenous lipids lipid, so they carry endogenous (CD1b-endo) lipids from the expression system. CD1b mutants and wild type control for experiments using these mutants, were produced in HEK293 S GnTI⁻ (American Type Culture Collection) and purified via HisTrap Metal chelating-Ni2⁺ affinity chromatography and size exclusion. For SPR, purified CD1b was loaded with a molar excess of C36 GMM (Bill and Melinda Gates Foundation lipid bank) over a 16 h incubation period at 20°C and purified by anion exchange chromatography. Recombinant PG10 and clone 2 TCRs were cloned into the pET30a vector and expressed in Escherichia coli BL21(DE3) as insoluble inclusion bodies, refolded in 20 mM Tris-HCl (pH 8.0), 5M urea, 400 mM L-arginine, 0.5 mM Na-EDTA, 5 mM reduced glutathione, and 0.5 mM oxidized glutathione for 3 days at 273 K, and purified.