| The isolation and solubilization procedure was performed essentially according to Sambrook et al. (1989). Cells lysis was done with lyzosyme (Sigma), and liberated DNA/RNA was digested with DNase I (Sigma) and RNAse A (Sigma). Inclusion bodies were collected by centrifugation at 17,000g for 10 min at 4°C and washed in PBS supplemented with 0.5 % (v/v) NP-40 (Sigma) and 0.1 % (w/v) deoxycholic acid (Sigma) followed by washing in 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM NaCl. Washed inclusion bodies were solubilized in 20 mM Tris-HCl (pH 8.0) and 8 M urea (200 mL for each 100 g of wet cell paste). Insoluble material was removed by centrifugation at 17,000g for 15 min at 4°C. Supernatants were pooled and successively filtered through 8-, 3-, 1.2-, and 0.45-µm filters and stored at -20°C until further processing.
Chromatographic separations were performed at 12°C on a ÄKTA prime workstation (Amersham Biosciences), using a flow rate of 60 mL/h. All chromatographic media and columns were from Amersham Biosciences, Sweden, including 1 mL HiTrap columns for screening experiments. The purification was monitored by SDS-PAGE and bicinchoninic acid (BCA) assay (Pierce). Urea decomposition leads to the generation of cyanate. To minimize the concentration of cyanate in urea-containing buffers, all solution were made freshly and used immediately, and the primary amine, Tris, was included as cyanate scavenger (Wingfield 2002). A preparation of solubilized inclusion bodies was applied to a Q Sepharose Fast Flow column (2.6 x 65 cm) equilibrated with 20 mM Tris-HCl (pH 8.0) and 8 M urea (Merck; loading buffer). Unbound material was washed off with 2 column volumes (CV) of loading buffer. Bound proteins were eluted with a linear 0 to 1 M NaCl gradient (5 CV). Fractions containing functional MHC-I heavy chain were pooled. Functional pools from Q Sepharose Fast Flow fractionation were adjusted to 100 mM Tris-HCl (pH 8.0) and 8 M urea, and ammonium sulfate (Sigma) was added to 20% (w/v) saturation. The solution was stirred at room temperature for 30 min, and insoluble material was removed by centrifugation at 17,000g for 10 min at 4°C. The supernatant was filtered through a 0.45-µm filter and applied to a phenyl Sepharose High Performance column (2.6 x 70 cm), equilibrated with 100 mM Tris-HCl (pH 8.0), 8 M urea, and 20% ammonium sulfate. Unbound proteins were washed off with 1.5 CV of the same buffer. Bound proteins were eluted from the column with a linear 20% to 0% ammonium sulfate gradient (5 CV). Prior to the subsequent size exclusion chromatography step, fractions were pooled and concentrated on a 10-kD nominal-molecular-weight-limit filter (Millipore) in a stirred nitrogen pressure cell (Amicon) to a final volume of 15 mL. Size exclusion chromatography was done on two Sephacryl 200-HR or two Sephacryl 400-HR columns (2.6 x 100 cm) connected in series. Columns were equilibrated with 20 mM Tris-HCl (pH 8.0), 8 M urea, or 20 mM Tris-HCl (pH 8.0), 6 M guanidine hydrochloride. Purified heavy chains were pooled, aliquoted, and stored at -20°C until further analysis.
Refolding conditions reported by Pedersen et al. (2001) for MHC-I heavy chain were used. Purified MHC-I heavy chain samples were refolded by 100-fold dilution in the presence of excess human ?m (3 µM) and radiolabeled peptide (1 to 3 nM, 15,000 cpm/sample) for 24 h at 18°C in a total reaction volume of 100 µL per sample. The refolding buffer was 100 mM Tris-maleate buffer (pH 6.6) in PBS supplemented with 1 mg/mL pluronic copolymer Lutrol F-68 (BASF). The final concentration of urea after dilution was 80 mM. The recombinant human ?m was produced in our laboratory from E. coli fermentations (Pedersen et al. 2001). Binding of peptide to MHC-I heavy chains were measured by Sephadex G-50 spun column chromatography (Buus et al. 1995). The radioactivity of the excluded `void` volume, containing formed MHC-I complexes, and of the retained volume, containing unbound peptide, was measured by gamma spectrometry (Packard Instruments). Peptide binding values were calculated by dividing excluded radioactivity with the total amount of radioactivity offered. Mean peptide binding values were obtained from duplicate spun column chromatography runs and expressed in percent.
Peptides were purchased from Schaefer-N, purified to homogeneity by reverse-phase HPLC chromatography, lyophilized, and stored at -20°C. All preparations were quantified by using the BCA assay. Radiolabeling was done with 125Iodine (Amersham Biosciences). Peptides used for refolding of MHC-I heavy chains, and biochemical binding assays had the following sequences (in single letter code): FLPSDYFPSV for HLA-A*0201, KLFPPLYLR for HLA-A*1101, and SDYEGRLI (Influenza NP peptide50?7) for H2-Kk (des cys). |