Endo h

PK2 laccase with α_OPT or α_{OPT Eα86T/Aα87N} were grown in 1.2 l EB medium using 1 l flasks as described above. After 4 days of incubation, liquid extracts were filtrated (through 0.22 μm cut off membrane) and concentrated and ultra-diafiltrated using Pellicon tangential filtration membranes (Merck Millipore, Germany) and Amicon stirred cells (Merck Millipore, Germany), both with a 10 kDa cut off. Laccases were purified by FPLC in three anion exchange and an exclusion size chromatography steps: (i) HiPrep QFF 16/10 column in a 100 ml gradient of 0–40% elution buffer, (ii) HiTrap QFF 5 ml in a 100 ml gradient of 0–40% elution buffer, (iii) Mono Q HR 5/5 column in a 30 ml gradient of 0–25% elution buffer, and (iv) Superdex 75. All columns were purchased from GE Healthcare. Enzyme purification was confirmed by SDS-PAGE (12% acrylamide) stained. For specific activity the final protein concentration was calculated by nanodrop (A280 nm) and laccase activity using 3 mM ABTS, 50 mM CP pH 3 in kinetic mode at 418 nm in SpectraMax M2 plate reader (Molecular Devices). Deglycosylation by Endo H (Merck) was performed following the seller´s recommendations.

The pattern of N-linked glycosylation of VLP-associated E2 was determined by treatment with PNGase F (Sigma) or Endo-H (Sigma). Samples containing 1 μg of protein were first denatured in 10 μL of buffer containing 0.5% (wt/vol) SDS and 40 mM dithiothreitol (DTT), with heating to 95°C for 10 min. High-mannose glycans were specifically removed using 1 unit of Endo-H in 50 μM sodium acetate (pH 6). All N-linked glycans were removed using 1 unit of PNGase F in 50 μM sodium phosphate (pH 7.5), containing 1% (vol/vol) NP-40. Untreated samples were also prepared in the absence of enzyme. Samples were then incubated at 37°C for 4 h before separation and detection via SDS-PAGE and Western blotting as described above.