Kta flux

To purify PPV VLPs by IEX chromatography, yeast cells were suspended in PBS buffer pH7.4 and disrupted by high-pressure homogenization. Cell lysates were subsequently adjusted to pH 4.0. After centrifugation at 10,000 rpm, 4 °C for 30 min, the supernatants of pH adjusted cell lysate were loaded onto an XK 50/30 column (GE Healthcare) packed with 400 mL of Capto S ImpAct resin. Binding VLPs were eluted with 20 mM sodium acetate buffer containing 500 mM NaCl. To elevate the recovery of VLPs, the precipitates of pH adjusted cell lysates were redissolved in an equal volume of 20 mM Tris-HCl buffer pH 8.0. Through centrifugation, the clarified supernatant were loaded onto an XK 50/30 column packed with 400 mL of Capto Q XP resins. After elution with 20 mM Tris-HCl buffer pH 8.0 plus 500 mM NaCl, fractions were diafiltrated for 10 volumes of PBS on ÄKTA flux (GE Healthcare, USA) equipped with a 750 kDa column (11-0005-50, GE healthcare). Further polishing purification of PPV VLPs was performed on an AKTA Purifier 100 (GE Healthcare, USA) using a HiPrep™ 26/60 Sephacryl® S-500 HR column (GE Healthcare). About 4 ml IEX purified sample was injected and eluted with PBS at a rate of 0.5 mL/min. Protein concentration was measured by the BCA Protein Assay Kit (23,250, Thermo Fisher Scientific).

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.

The filtered supernatant was concentrated ~ 20-fold using tangential flow filtration (Äkta flux, GE Healthcare) with a nominal molecular weight cut-off of 100 kDa (UFP-100-C-3X2MA, Cytiva) and ultracentrifuged afterward at 100,000 × g for 1 h and 4 °C. The pellet was washed in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.25) and the supernatant was discarded. Subsequently, the MVs were purified by density gradient centrifugation with OptiPrep (60% Iodixanol). The MV containing pellet was resuspended in 2 ml 50% Iodixanol, diluted with a solution of 0.85% NaCl and 60 mM HEPES (pH 7.4). Then, carefully, 1 ml of 40%, 30%, and 10% Iodixanol diluted with 0.85% NaCl and 10 mM HEPES (pH 7.4) were carefully layered from the highest to the lowest Iodixanol concentration above the 50% solution layer. The samples were ultracentrifuged in a swing-out rotor at 100,000 × g for 2 h at 4 °C. The third and fourth 1 ml fractions contained the MVs and were transferred into new ultracentrifugation tubes, diluted five to sixfold with TE buffer, and ultracentrifuged again. Finally, the pellets of both fractions were pooled in TE buffer, transferred into a new reaction tube, and ultracentrifuged again. The MV containing pellet is referred to as “MV fraction” in this study and was stored at − 20 °C.