Perfectsil 300 ods c18

The high-affinity HLA-A*03:01 binder AIFQSSMTK (K_D ≈ 10 nM, HIV-derived epitope) was chosen to create the CSN peptide (AIFCSNMTK, K_D ≈ 10 nM) for fluorescent labeling. Peptide affinities were predicted with NetMHC 4.1 (ref. 34 (link)). Peptides were synthesized by automated microwave-assisted solid-phase peptide synthesis by Fmoc chemistry (CEM, Liberty Blue). The CSN peptide and C4 peptide (RRYCKSTEL, K_D ≈ 20 µM) were labeled with 5-iodoacetamide fluorescein (5-IAF, Sigma–Aldrich) in PBS/DMF buffer (8.1 mM Na₂HPO₄ pH 6.5, 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 33% (v/v) DMF) with heavy agitation for 2 h at 20 °C using a two-fold molar excess of 5-IAF. Samples were purified by reversed phase C₁₈ HPLC (Agilent, 1200 Series System; PerfectSil 300 ODS C₁₈ 5 µm 300 × 10 mm) applying a linear water/acetonitrile gradient from 5–60% supplemented with 0.1% (v/v) TFA. Purified peptides were snap frozen in liquid nitrogen and lyophilized (Lyovac GT2, Heraeus). Peptide identity was confirmed by LC-MS analysis (AIFCSNMTK: [M + H⁺]_calc: 1014.475 Da, [M + H⁺]_obs: 1014.475 Da; AIFC^FSNMTK: [M + H⁺]_calc: 1401.550 Da, [M + H⁺]_obs: 1401.549 Da; RRYQKSTEL: [M + H⁺]_calc: 1180.636 Da, [M + H⁺]_obs: 1180.635 Da; RRYC^FKSTEL: [M + H⁺]_calc: 1542.669 Da, [M + H⁺]_obs: 1542.663 Da).

The ICP47^SBP sequence was cloned into a pETM-11 (European Molecular Biology Laboratory, EMBL) using the NcoI and BamHI restriction sites. Escherichia coli One Shot BL21(DE3) cells (Thermo Fisher) were transformed and grown to OD₆₀₀ = 0.6 at 37 °C. Expression was induced with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) for 6 h at 22 °C. The cells were lysed by sonication in 50 mM NaP_i pH 8.0, 150 mM NaCl, 20 mM imidazole, 0.2% Tween 20, 2 mM DTT, supplemented with 2.5 mM PMSF, 3.75 mg/mL lysozyme, 6.25 mM benzamidine, and 1 U/mL benzonase. His₆-TEV-ICP47^SBP was isolated from the lysate via reverse IMAC (Ni-NTA Agarose resin, QIAGEN). After washing with 50 mM NaP_i pH 8.0, 150 mM NaCl, 20 mM imidazole, 0.2% Tween 20, 10 mM DTT, ICP47^SBP was eluted supplementing the wash buffer with 500 mM imidazole. The His₆-tag was removed by overnight TEV protease digestion. ICP47^SBP was purified by reversed phase C₁₈ HPLC (Agilent, 1200 Series System; PerfectSil 300 ODS C₁₈ 5 µm 300x10 mm) applying a linear water/acetonitrile gradient from 5–60% supplemented with 0.1% (v/v) TFA. The identity of ICP47^SBP was confirmed by LC-MS analysis (M_calc: 14694.06 Da, M_obs: 14694.75 Da).