Kimata, Y., Ishiwata-Kimata, Y., Ito, T., Hirata, A., Suzuki, T., Oikawa, D., Takeuchi, M. and Kohno, K.
Notes: The authors examined the mechanism by which the signal transducer Ire1 senses the accumulation of unfolded proteins during endoplasmic reticulum stress. The authors tested the hypothesis that the core stress-sensing region (CSSR) of Ire1 binds to unfolded proteins by monitoring the ability of purified CSSR and mutant CSSRs to prevent aggregation of denatured firefly luciferase and porcine citrate synthase. CSSR and CSSR mutants were expressed as maltose-binding protein fusions or His8-tagged proteins; the His8-CSSR was expressed in BL21(DE3) cells and purified using HisLink™ Protein Purification Resin. Cell pellets from 400ml cultures were resuspended in 15ml of E. coli lysis buffer (50mM Hepes, pH 8.0, 300mM KCl,
5mM MgCl2, 10mM imidazole, 1% Triton X-100, 2mM phenylmethylsulfonyl fluoride, 0.4mg/ml benzamidine, 0.4mg/ml pepstatin A, 0.4mg/ml leupeptin, 0.3mg/ml lysozyme and 14U/ml DNase I), then disrupted by ultrasonication. Lysates were clarified by centrifugation and incubated with 0.5ml of HisLink™ Resin for 12 hours. The resin was packed into an 8mm-diameter column and sequentially washed with 1) 50mM Hepes (pH 8.0), 1M KCl, 5mM MgCl2, and 0.1% Triton-X 100; 2) 50mM Hepes, 300mM KCl, 5mM MgCl2, 0.1% Triton X-100, and 20mM imidazole; 3) 50mM Hepes, 300mM KCl, 5mM MgCl2, 0.1% Triton X-100, and 40mM imidazole; 4) 50mM Hepes, 300mM KCl, 5mM MgCl2, 0.1% Triton X-100, and 60mM imidazole; 5) 50mM Hepes, 300mM KCl, 5mM MgCl2, and 10mM ATP; and finally 20mM Hepes, 100mM KCl, 5mM MgCl2, and 50% (vol/vol) glycerol. Bound proteins were eluted with 50mM Hepes, 100mM KCl, 5mM MgCl2, 200mM imidazole and 50% (vol/vol) glycerol. (3914)