Triton x 100

The transformation of the T. reesei WT was performed as described by Gruber and coworkers [23 (link)]. A total of 20 μg of linear DNA from the deletion cassette was used in the assay. After transformation, the plates were incubated at 30 °C for 3 to 4 days until spores were visible. The candidates were submitted to three rounds of selection on MA medium with and without 0.1% Triton X-100 (GE Healthcare, Uppsala, Sweden). Cassette integration in the genome locus was verified by PCR using a specific set of primers for pyr4 and epl2 (Table S2). The expression profile of the epl2 gene was also analyzed by RT-qPCR (Table S3).

Protein extracts were prepared in biological triplicate (300 mg FM) for each maturation treatment. Soluble proteins were extracted according to the method described by Santa-Catarina et al. [110 ]. The buffer-soluble proteins were extracted with phosphate buffer (pH 7.5) containing 50 mM sodium phosphate dibasic (Vetec), 10 mM 2-mercaptoethanol (Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich). The supernatants were transferred to clear microtubes, and the proteins were precipitated on ice for 30 min with 10% trichloroacetic acid (Merck). The pellet was washed three times with cold acetone (Merck), and the proteins were then resuspended and concentrated in 0.5 mL of a urea/thiourea buffer (7 M urea, 2 M thiourea, 1% dithiothreitol (DTT), 2% Triton X-100, 0.5% pharmalyte (all from GE Healthcare, Freiburg, Germany) and 1 mM PMSF), to which a 0.5% immobilized pH gradient (IPG) buffer (pH 4–7) (GE Healthcare) was added. The protein concentration was determined using the 2-D Quant Kit (GE Healthcare).

The purification procedure is similar as in^{47 (link)} with the following modifications: 1) The membrane fraction was solubilized by addition of an equal volume of: 10 mM sodium phosphate (NaP_i) at pH 8, 10% glycerol, 0.1 mM EDTA, 1 mM GSH, and 6% Triton X-100 (Sigma) and followed by 45 minutes incubation in 4 °C; 2) The hydroxyapatite affinity chromatography was replaced by immobilized metal ion affinity chromatography (IMAC), (column: Hitrap chelating, from GE healthcare). The unspecific proteins were washed in 10 mM NaP_i at pH 8, 150 mM NaCl, 1 mM GSH, 10% glycerol, 50 mM imidazole, and 0.1% reduced Triton X-100 (Sigma) and rat MGST1 protein was eluted in 350 mM imidazole; 3) After IMAC, the eluted peak was immediately desalted (column: HiPrep 26/10, from GE healthcare) in buffer A: 10 mM NaP_i at pH 8, 30 mM NaCl, 1 mM GSH, 10% glycerol, and 0.1% reduced Triton X-100. The desalted sample was further purified by cation exchange chromatography (column: Hitrap SP, from GE healthcare) equilibrated with buffer A. Rat MGST1 protein was eluted with 300 mM NaCl in buffer A. The pooled fractions (approximate 6 ml) were concentrated (approximate 0.6 ml) (centrifugal tube: Amicon Ultra-4 centrifugal filter unit with 10 kDa cutoff, from Millipore) and followed by 2D crystallization trials. The activity measurement was performed according to^{49 (link)}.