M 110l microfluidizer

Constructs of pET24sinR^WT and their mutagenized variants were transformed into E. coli BL21(DE3) for protein overexpression. Proteins were overexpressed in 1 l LB autoinduction media (1.8% w/v lactose) shaking at 30 °C overnight. Cells were harvested the next morning and resuspended in 20 ml buffer A (20 mM HEPES/NaOH pH 8.0, 500 mM NaCl, 40 mM imidazole). Cells were lysed two times with a M-110 L Microfluidizer (Microfluidics) and centrifuged at 20,000 r.p.m. for 20 min at 4 °C to remove cell debris. The supernatant was applied onto a 1 ml HisTrap HP column (GE Healthcare) for Ni-NTA affinity chromatography. The column was washed with 15 column volumes of buffer A and proteins were eluted with 5 ml buffer B (20 mM HEPES/NaOH, pH 8.0, 500 mM NaCl, 500 mM imidazole). Proteins were concentrated to 1 ml and further purified by size-exclusion chromatography using a HiLoad 26/60 Superdex 200 gel-filtration column in buffer C (20 mM HEPES/NaOH, 500 mM NaCl).

Protein overexpression was performed as described previously (25 (link)) with the following modifications of the protein purification protocol. Cell pellets were resuspended in 50 ml of buffer A (20 mM Tris HCl [pH 8.0], 200 mM NaCl, 10% glycerol, and 1 mM tris-2-carboxyethyl phosphine hydrochloride [TCEP]) and lysed by an M-110L microfluidizer (Microfluidics). The protein was purified from the clarified cell lysate by Ni-nitrilotriacetic acid (NTA) affinity chromatography to >95% homogeneity. The hexahistidine-tagged MtrR was cleaved by thrombin (GE Healthcare) digestion, and the cleaved MtrR was purified from tagged MtrR and the cleaved hexahistidine tag by Ni-NTA affinity chromatography. Further purification was performed by size exclusion chromatography (S200 column), and the purified protein was concentrated to ∼30 mg/ml using an Amicon YM-10 membrane filter. Semet-MtrR was overexpressed using the methionine-inhibitory pathway (48 (link)) and purified as described for native MtrR.

The expression cultures were harvested by centrifugation (6000g for 15 min at 293 K) and the pellets were resuspended in 10 ml lysis buffer per gram of cells and processed using an M-110L Microfluidizer (Microfluidics). The lysis buffer consisted of 20 mM Tris pH 8.5, 500 mM NaCl, 10 mM MgCl₂, 10 mM KCl, 10%(v/v) glycerol. The lysate underwent clarification by centrifugation (125 000g for 30 min at 293 K) using a Ti-45 rotor (Beckmann) and was applied onto a 1 ml HisTrap HP column (GE Healthcare). The column underwent an initial wash with five column volumes of lysis buffer containing 40 mM imidazole pH 8.5. Protein elution took place in lysis buffer containing 500 mM imidazole pH 8.5. Subsequently, the protein was concentrated to approximately 30 mg ml⁻¹ using an Amicon Ultracel-10K (Millipore) and was subjected to size-exclusion chromatography using an Superdex 75 XK 26/600 column (GE Healthcare) in the same buffer as above but without imidazole. Protein-containing fractions were combined and concentrated to 10 mg ml⁻¹.

Overexpression and purification of PglB was performed as previously described^{19 (link)}. Briefly, PglB was overexpressed in Escherichia coli BL21-Gold cells (DE3) (Strategene) at 37 °C in five-liter flasks using Terrific Broth medium supplemented with 1% glycerol (w/v). The cells were induced at 37 °C for 4 h at A600 of 3.0 by adding 0.1% arabinose (w/v). All following steps were carried out at 4 °C. Cells were harvested by centrifugation. Cells were resuspended in 25 mM Tris-HCl, pH 8.0, 250 mM NaCl and disrupted in a M-110-l microfluidizer (Microfluidics) at 15,000 p.s.i. chamber pressure. Membranes were pelleted by centrifugation at 100,000 g for 30 min and solubilized in 25 mM Tris-HCl, pH 8.0, 250 mM NaCl, 10% glycerol (v/v) and 1% N-dodecyl-β-D-maltopyranoside (w/v) (DDM, Anatrace) for 1.5 h. All subsequent purification buffers contained 0.016% DDM. PglB was purified on a nickel-nitrilotriacetic acid (Ni-NTA) Superflow affinity column (Qiagen) and desalted into 10 mM MES, pH 6.5, 100 mM NaCl, 3% glycerol (v/v), and 0.016% DDM. For crystallization experiments, desalted protein was concentrated to 6 mg mL⁻¹ in an Amicon Ultra-15 concentrator (Milipore) with a molecular mass cutoff of 100 kDa and purified by size exclusion chromatography (Superdex 200 10/300, GE Healthcare) in desalting buffer. Collected fractions were pooled and concentrated to 12 mg mL⁻¹.

Harvested cell pellets were resuspended (50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0) and lysed by high-pressure homogenization (M-110L Microfluidizer, Microfluidics). After centrifugation (18,000 g, 60 min, 4°C), cleared lysates were loaded onto equilibrated Ni-NTA columns and purified via a peristaltic pump (Pharmacia Biotech, Stockholm, SE). After washing with 10 column volumes of resuspension buffer, elution buffer (50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 8.0) was applied to elute the his-tagged target proteins. The first 2 ml (covering the dead volume) were discarded. The eluate was collected in 2 ml fractions. After taking SDS samples, fractions were pooled and dialyzed in cellulose film tubings against 1 L buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0) for at least 1.5 h with three buffer changes. Concentrations of purified reporter proteins were determined by measuring the sfGFP chromophore absorption at 488 nm.

Cloning of Utp4 from Chaetomium thermophilum was described previously [12 (link)]. Expression of native Utp4 from Chaetomium thermophilum (Uniprot CTHT_0058380, residues 1–848) was carried out in E. coli Rosetta 2 (DE3) cells in ZYM5052 medium, grown at 37°C to an OD₆₀₀ of 1.0. Then, the temperature was dropped to 23°C and the cells were grown further overnight. After harvesting, the cell pellet was mixed with lysis buffer (20 mM HEPES pH 8.0, 250 mM NaCl, 20 mM MgCl₂, 20 mM KCl, 40 mM imidazole) and lysed in a M-110L Microfluidizer (Microfluidics). Lysate was clarified by centrifugation (20000 ×g, 20 min, 277 K) and the filtered supernatant was loaded onto a 5 ml HisTrap FF column (GE Healthcare) previously equilibrated with 10 column volumes (CV) of lysis buffer. The column was further washed with 10 CV of lysis buffer and the protein was eluted using 5 CV of wash buffer containing 500 mM imidazole. Elution fractions containing Utp4 were concentrated and further purified by size exclusion chromatography (SEC). The sample was loaded onto a Superdex 200 26/60 prep grade column (GE Healthcare) equilibrated with SEC buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 20 mM MgCl₂, 20 mM KCl, 1 mM DTT). Se-Met labelled Utp4 was expressed as the native protein using a modified protocol for the inhibition of methionine-biosynthesis with 0.5 mM IPTG induction [13 (link)].

His₆-RgsD was purified as described previously (57 (link)). Briefly, cells were lysed using M110-L microfluidizer (Microfluidics), and the protein was isolated using a 1-ml HisTrap column (GE Healthcare). Protein was concentrated with Amicon Ultracel-10K (Millipore) to a volume of 2 ml and applied to size exclusion chromatography (SEC) (HiLoad 26/600 Superdex 200 pg; GE Healthcare). After SEC, protein-containing fractions were pooled and concentrated with an Amicon Ultracel-10K (Millipore) according to the experimental requirements.