Ni nta superflow column

Large scale Rac1 expression was conducted according to the established protocol described above. Sf9 insect cells were inoculated at a density of 1.5×10⁶ cells/ml under optimized MOI and culture time. Cells were resuspended in lysis buffer, containing 50 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 2 mM β-mercaptoethanol, 5 mM MgCl₂, 0.1 mM GDP, 10 mM imidazole and the optimized detergents according to the screening procedure described above. Cells were disrupted by sonication in ice-water mixture. Supernatants were collected by centrifugation and loaded on a Ni-NTA Superflow column (Qiagen, Hilden, Germany). High salt buffer (50 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 2 mM β-mercaptoethanol, 5 mM MgCl₂, 0.1 mM GDP, 10 mM imidazole, 350 mM KCl and 1 mM ATP) was used to remove impurities from the target proteins. Rac1 protein was eluted using an imidazole gradient ranging from 10 to 500 mM. The protein solution was concentrated and further purified on a Superdex 75 column (10/300 GL, GE-Healthcare, Uppsala, Sweden) with 50 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 3 mM DTT, 5 mM MgCl₂ and 0.5% (w/v) Na-cholate as buffer system. GppNHp-bound Rac1 proteins as well as human GDI1, RacGEFs, Pak1, full length and C-terminal truncated Rac1 proteins were prepared from E. coli as described previously [11] (link), [43] (link).

The cells expressing recombinant TanA_Lb were resuspended in lysis buffer [20 mM Tris-HCl, 20 mM imidazole, 500 mM NaCl, 1 mM dithiothreitol (DTT), pH 8.0] and ultrasonicated at 1.5-s pulse and 3-s output for 15 min. Then, the crude lysate was centrifuged at 40,000 × g and 4°C for 30 min, and the supernatant was bound to a 1 ml of Ni-NTA Superflow column (Qiagen, Hilden, Germany). After washing with 10 ml of wash buffer (20 mM Tris-HCl, 0.5 M NaCl, 1 mM DTT, 50 mM imidazole, pH 8.0), the TanA_Lb protein was eluted with 10 ml of elution buffer (20 mM Tris-HCl, 0.3 M NaCl, 1 mM DTT, 0.5 M imidazole, pH 8.0). For further purification, the target protein was buffer exchanged into 20 mM Tris–HCl pH 8.0, 1 mM DTT, loaded onto a 5-ml MonoQ ion exchange column (GE Healthcare, Piscataway, NJ, USA), and eluted using a linear gradient using from 0 to 1 M NaCl. Then, gel filtration chromatography was used to determine the apparent mass of the TanA_Lb protein using a Superdex 200 increase column (GE Healthcare, Piscataway, NJ, USA). The running buffer was 20 mM Tris-HCl (pH 8.0, containing 150 mM NaCl and 1 mM DTT), and the flow rate was 0.5 ml/min. Finally, the purified protein was stored at 4°C until further analysis. The molecular mass and purity of the target protein were assessed by SDS-PAGE.

PCR was performed to add NheI and SacI restriction sites to Vd2LysM. Using directional cloning, Vd2LysM was cloned into vector pHYG, a modified version of the expression vector pMDC32 (Curtis and Grossniklaus, 2003). The expression vector was subsequently transferred into Agrobacterium tumefaciens strain MOG101. Infiltration of 4–5‐week‐old N. benthamiana with Agrobacterium tumefaciens was performed as described by Westerhof et al. (2012). Four days after infiltration, leaves were harvested and flash frozen in liquid nitrogen. Plant material was ground and homogenized in ice‐cold extraction buffer [1% v/v Tween‐20, 2% w/v immobilized polyvinylpolypyrrolidone (PVPP), 300 mm NaCl, 50 mm NaH₂PO₄, 10 mm imidazole, 1 mm dithiothreitol (DTT), pH 7.4] using 2 mL/g fresh plant material. After 30 min of homogenization at 4°C, the crude extract was centrifuged at 16 000 g at 4°C. The supernatant was further cleaned using a miracloth filter, and an Ni‐NTA Superflow column (Qiagen) was used to purify the HIS‐tagged protein, according to the manufacturer's protocol. The final protein concentration was determined spectrophotometrically at 280 nm and confirmed using the Pierce BCA Protein Assay Kit (Thermo Scientific) with BSA as a standard.