Deae sepharose

Site‐directed mutagenesis was performed with the use of non‐overlapping primers (Table S4) according to Q5 protocol (New England Biolabs). As a PCR matrix, the EcAIII gene cloned to the pET11d vector was used. The presence of mutations in the EcAIII gene was confirmed by sequencing (Genomed, Poland). Large‐scale expression was performed in 1 L of liquid LB medium supplemented with 100 μg/mL ampicillin. Cells were grown at 37°C until the OD₆₀₀ reached 1.00. The bacterial cultures were cooled to 4°C (30 min) and protein expression was induced with 0.5 mM IPTG. Protein production was carried out overnight at 18°C. Cell pellets were resuspended in a 20 mM Tris–HCl pH 8.0 buffer. Cell lysis was performed by sonication. Clarified cell lysate was loaded on an ion‐exchange column (DEAE‐Sepharose, Merck) connected to an ÄKTA FPLC system (GH Healthcare). Fractions were eluted from the column using a gradient of 2 M NaCl in 20 mM Tris–HCl pH 8.0. Fractions were analyzed by SDS‐PAGE and those containing the overexpressed protein were pooled, concentrated, and loaded on a size‐exclusion chromatography (SEC) column (Sephadex G75, GE Healthcare). Fractions were eluted from the SEC column using 100 mM Tris–HCl pH 8.0 with 200 mM NaCl.

The supernatant containing myrosinase was pre-filtered using a wine filter and subjected to microfiltration on 0.2 μm membranes (Hydrosart^® Microfilter, Sartorius, Göttingen, Germany) using an ÄKTA flux (GE Healthcare, Chicago, IL, USA). The cell-free supernatant was desalted and concentrated 10-fold with a 30 kDa cut-off membrane (TANGEN XTM PRO PDn Cassette, ProStream, REPLIGEN, Waltham, MA, USA) with 50 mM sodium phosphate buffer, pH 6.5. A 5 mL aliquot of the desalted supernatant was diluted with MilliQ water to 30 mL and the pH was adjusted to 8 with 2 M NaOH. The supernatant was loaded onto a column (1.1 × 11.5 cm, flow 2.5 mL/min) of DEAE-Sepharose (Merck, Darmstadt, Germany) equilibrated with 25 mM Tris-HCl buffer, pH 8 (buffer A) [modified from Härtel and Brandt [22 (link)]]. Three concentrations of buffer B (250 mM NaCl in buffer A) were used to elute proteins from the column. Contaminating proteins were eluted from the column at 35% of buffer B (87.5 mM NaCl) for 8 CV, myrosinase was eluted at 60% of buffer B (150 mM NaCl) for 8 CV, and the most tightly bound proteins were eluted at 100% of buffer B. The myrosinase-containing fractions were concentrated and desalted with Amicon^® Ultra Centrifugal Filters, 30 kDa cut-off (Merck Darmstadt, Germany) and stored at −80 °C without glycerol.

Starting cultures were transferred into 1 l LB medium and the cells were grown at 37°C until the OD₆₀₀ was close to 1.00. Next, the cultures were cooled to 4°C for 30 min and protein expression was induced using 0.5 mM IPTG. Expression was carried out overnight at 18°C. The cells were harvested by centrifugation. The cell pellets were resuspended in 20 mM Tris–HCl pH 8.0 buffer and cell lysis was performed by sonication followed by centrifugation. The clarified cell lysate was fractionated by ion-exchange chromatography (DEAE-Sepharose, Merck). Fractions were eluted from the column using a gradient of 2 M NaCl in 20 mM Tris–HCl pH 8.0. Fractions were analyzed by SDS–PAGE and those containing the desired protein were pooled, concentrated and loaded onto a size-exclusion chromatography (SEC) column (Sephadex G75, GE Healthcare) connected to an ÄKTA FPLC chromatography system (GE Healthcare).