Seeblue

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed using an analysis kit with pre-cast gradient mini-gels (Invitrogen Novex Bolt, 4–12%, Bis-Tris Plus, Thermo Fisher Scientific, Waltham, MA, USA), and a protein ladder (Invitrogen SeeBlue^®). A voltage of 145 V was applied during 35 min for separation. The protein bands were stained for 15 min (GelCode^TM Blue Safe protein stain, Thermo Scientific, USA), and the gels were washed overnight. For SDS-PAGE analysis of freeze-dried protein concentrates, the material was dissolved in MilliQ water to reach a concentration of 4 mg dry material/mL and the samples were mixed at room temperature for 10 min before analysis. In SDS-PAGE analysis, the RuBisCO subunits are found at ~55 kDa and ~14 kDa [10 (link)].

The P. digitatum AfpB was produced and purified as previously described (Hernanz-Koers et al., 2018 (link)). PeAfpA was purified from a 10-day PcMM supernatant of P. expansum CMP-1 strain. PeAfpB and PeAfpC were purified from supernatants of P. chrysogenum transformant strains growing in PcMM for 72–96 h. Cell-free supernatant containing PeAfpA was dialyzed (2 K MWCO, Sigma-Aldrich) against 20 mM phosphate buffer pH 6.6, and supernatants containing PeAfpB and PeAfpC were dialyzed against 20 mM acetate buffer pH 5.4. Dialyzed solutions were applied to an AKTA Purifier system equipped with 6 mL RESOURCE^TM S column (GE Healthcare Life Sciences, Little Chalfont, United Kingdom) equilibrated in the corresponding buffer. Proteins were eluted applying a linear gradient from 0 to 1 M NaCl in the same buffer.
Protein containing fractions were pooled, dialyzed against Milli-Q water, and lyophilized. Protein concentrations were determined by spectrophotometry (A₂₈₀) considering their molar extinction coefficients (𝜀₂₈₀ = 0.64 for PeAfpA, and 𝜀₂₈₀ = 0.67 for PeAfpB and PeAfpC). The purity was checked by SDS-PAGE (Laemmli, 1970 (link)) using SDS-16% polyacrylamide gels calibrated with prestained protein size-standard SeeBlue^® (Thermo Fisher Scientific, Waltham, MA, United States) and Coomassie blue stained.

Culture supernatant and recombinant proteins (AAT (Athens Research), SLPI (R&D Systems), elafin (Proteo Biotech)) were quantified by bicinchoninic acid assay and electrophoresed on 12.5% SDS-polyacrylamide gels. For band size determination the protein marker SeeBlue® (Thermo Fisher Scientific: Waltham, MA, USA) Pre-stained Protein Standard (Invitrogen, LC5625) (Thermo Fisher Scientific: Waltham, MA, USA) or Mark12^TM Unstained Standard (Invitrogen, Bio Sciences Ltd, Ireland) was used. For Coomassie blue silver staining, gels were fixed in ethanol (50% v/v) and phosphoric acid (2%) for one hour and washed × 3 in dH2O prior to staining with Coomassie blue silver overnight. For western blotting, following SDS-PAGE, proteins were transferred to PVDF and signals were detected using goat anti-AAT (Abcam) (https://www.biocompare.com/), goat anti-SLPI (R&D Systems) or mouse anti-elafin (Abcam) with appropriate secondary antibodies and Immobilon Western chemiluminescent HRP substrate (Millipore) using the Syngene G:Box Chemi XL gel documentation system (Syngene International Limited, New Delhi, India).

SDS-PAGE on 4–12% NuPage gels (Life Technologies) was performed for labelled and unlabelled protein/Abs samples. Loading dye was added to each sample and heated at 100 °C for 5 minutes. SeeBlue™ (ThermoFisher Scientific) was used as a ladder. Lanes were loaded at similar protein concentrations and the gel was run with MES buffer at 180 V for 40 minutes. The images were acquired after staining with Pageblue (ThermoFisher Scientific/Pierce) protein staining solution.

AfpB variants were purified from the supernatants of P. digitatum transformant strains (AfpB and AfpB*) and P. pastoris positive transformants (PpAfpB*). Cell-free supernatants of P. digitatum grown on PdMM for 11 days or P. pastoris grown on BMM for 2 days were collected by centrifugation and dialyzed (2 K MWCO,) against 20 mM phosphate buffer pH 6.6. Dialyzed solutions were applied to an AKTA Purifier system equipped with a 6 ml RESOURCE S column (GE Healthcare) for P. digitatum supernatants or a 5 ml HiTrap SP HP column (GE Healthcare) for P. pastoris supernatants, equilibrated in phosphate buffer. Proteins were eluted with a linear NaCl gradient from 0 to 0.5 M in the same buffer.
Protein containing fractions were pooled, dialyzed against Milli-Q water and lyophilized. Protein concentrations were determined spectrophotometrically (A₂₈₀) considering the molar extinction coefficient (ε280 = 0.52). Purification was monitored by SDS-PAGE^{44 (link)} using SDS-16% polyacrylamide gels calibrated with prestained protein size-standard SeeBlue® (ThermoFischer Scientific) or Precision Plus Protein Standards (Bio-Rad) and Coomassie stained.