― 252 ―resin column (IBA Lifesciences). After elution with buffer G supplemented with 50 mM biotin, the eluate was subjected to size-exclusion chromatography using HiLoad 26/600 Superdex 200 prep grade (Cytiva) and eluted with 20 mM Tris-HCl (pH 8.0) and 200 mM NaCl. The purified Atg18 was concentrated, supplemented with 1 mM DTT, and stored at -80°C until use.Expression and purification of Nur proteinThe pGBHPS-Nur-YC plasmid was transformed into E. coli BL21 (DE3) to express Nur lacking its signal peptide. A single tyrosine and cysteine residue were inserted at the C-terminus for quantification and modification, respectively. After production of the protein in E. coli, cell pellets were resuspended in 30 mL wash buffer (500 mM NaCl, 10 mM imidazole, 50 mM Tris-HCl, pH 8.0) and boiled for 25 minutes. After centrifugation, the supernatant was mixed with 5 mL Ni-NTA agarose resin (QIAGEN) and applied to a gravity-flow column. The column was washed with 50 mL of wash buffer, and bound proteins were eluted with 10 mL of elution buffer (500 mM NaCl, 200 mM imidazole, 50 mM Tris-HCl, pH 8.0). The His-tag was removed by overnight incubation with 10–20 μL of HRV3C Protease at 4°C. Further purification was performed using a Mono S 5/50 GL cation exchange column (GE Healthcare) on an AKTA system at 4°C.Small unilamellar vesicles (SUVs) preparationSUVs were prepared using an ultrasonic method. The lipid composition of donor SUVs for lipid transfer assay was DOPC:DOPE:DOPS:cy5.5-PE = 68:15:12:5. Each lipid was dissolved in chloroform, dried under a stream of nitrogen gas, and subsequently subjected to vacuum drying for over 4 h. The dried lipids were then resuspended in buffer H [20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1 mM DTT] to a final concentration of 1 mM. The suspension was sonicated (output 8) for 30 s followed by incubation on ice, and this cycle was repeated 10 times to ensure proper formation of SUVs.Giant unilamellar vesicles (GUVs) preparation and experimentGUVs were prepared by the natural swelling method. DOPC, DOPE, DOPS, and PI3P were used for GUVs for the Atg2 project, whereas POPG and POPC were used for GUVs for the Nur project. A total of 2.0 μmol of dry lipid film was hydrated with 20 μL of water in a glass vial and incubated at 45°C for 7 minutes. The pre-hydrated lipid was then added to 1 mL of buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM egtazic acid, and 0.10 M sucrose, followed by incubation for 2–3 hours at 37°C. The supernatants were collected by centrifugation at 13,000 ×g for 20 minutes at 20°C and passed through a nucleopore membrane with 10 μm diameter pores (GE Healthcare, Whatman) using buffer containing 0.10 M glucose at a flow rate of 1 mL/min for 1 hour at room temperature. The GUVs retained on the filter were collected as purified vesicles. Purified GUVs were transferred to microchambers pre-coated with 0.10% w/v BSA and observed under a confocal laser scanning microscope (Olympus, FV 3000) equipped with a ×60 objective lens. Fluorescently labeled proteins were applied using a glass micropipette with a 20 μm diameter tip, positioned approximately 60 μm from the target GUV. The pressure difference was adjusted to −30 Pa and monitored using a differential pressure
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