GMCBM21, GM64–237, GM2–237, and GM64–237 N228A cell pellets were resuspended in ice-cold lysis buffer [50 mM tris (pH 8.0), 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, and an EDTA-free protease inhibitor tablet (Roche)] and lysed by high-pressure homogenization (Avestin EmulsiFlex C3). Lysate was clarified by centrifugation (45,000g, 45 min, 4°C), and the supernatant was loaded onto a HisTrap column (GE Healthcare) pre-equilibrated with 50 mM tris (pH 8.0), 500 mM NaCl, and 5 mM imidazole. Protein was eluted using a gradient of 5 to 500 mM imidazole. Fractions containing GMCBM21 were pooled and dialyzed overnight at 4°C against 50 mM tris (pH 8.0), 500 mM NaCl, and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) to cleave the His6-tag. The cleaved protein was incubated with the Ni+2-NTA (nitrilotriacetic acid) beads (GE Healthcare) to remove the TEV protease and cleaved His-tag. The flow-through was collected, concentrated, and further purified using size exclusion chromatography [SEC; Superdex 75 26/60 (GE Healthcare)] equilibrated in NMR buffer [20 mM sodium phosphate (pH 6.5), 50 mM NaCl, and 10 mM dithiothreitol (DTT) or 20 mM bis-tris (pH 6.8), 150 mM NaCl, and 0.5 mM TCEP] or ITC buffer [20 mM tris (pH 8.0), 0.5 M NaCl, 0.5 mM TCEP, and 1 mM MnCl2]. Fractions were pooled, concentrated, and stored at −20°C. 15N-labeled GM64–93 was purified identically except that the protein was heat purified at 95°C (15 min), and the supernatant was collected and concentrated before SEC. PP1 was purified as previously described (18).

The GST-GYG1:GYS1 complex was purified using glutathione agarose beads (Pierce). The Sf9 cell pellet (8 g) was lysed in ice-cold lysis buffer [50 mM tris (pH 8.0), 150 mM NaCl, 5% glycerol, 1 mM EDTA, and 0.1% Triton X-100] with cOmplete mini protease inhibitor cocktail tablets (Roche). Lysate was clarified by centrifugation (40,000g, 45 min) and filtered through a 0.22-μm syringe filter. The supernatant was incubated on a rolling platform for 1 hour at 4°C with 1-ml bed volume of glutathione agarose resin pre-equilibrated in low-salt buffer [50 mM tris (pH 8.0), 150 mM NaCl, 5% glycerol, and 1 mM EDTA]. The beads were then washed with 10 column volumes (CVs) of low-salt buffer, followed by 50 CVs of high-salt buffer [50 mM tris (pH 8.0), 500 mM NaCl, 5% glycerol, and 1 mM EDTA], and followed again by 10 CVs of low-salt buffer. The complex was eluted with 10 mM fresh reduced glutathione, concentrated to 1 mg/ml, flash frozen in liquid nitrogen, and stored at −80°C until needed.

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