Akta start

Escherichia coli BL21 bacteria harboring pET28a-bcnA were grown for 3 h with 500 mM isopropyl thio-β-galactoside (ThermoFisher Scientific, United Kingdom) at 25°C; bacteria were harvested, resuspended in 50 mM phosphate buffer (PBS), pH 7.4 (Sigma-Aldrich, United Kingdom), with protease inhibitors (Roche Diagnostic GmBH, Germany), and lysed using a cell disruptor at 27 kpsi (Constant Systems). The lysate was cleared by centrifugation at 16,000 × g for 60 min at 4°C (Sorvall RC 6 Plus, Germany), and the supernatant was filtered through 0.2-μm in-line filters. The supernatant was passed through a HisTrap FF 5-ml column, using AKTA Start (GE Healthcare Bio-science, Sweden). The purified protein was detected by Coomassie blue staining following 16% SDS-PAGE and quantified by Bradford assay using bovine serum albumin (BSA) as standard.

To isolate extracellular vesicles (EV), initially we collected serum-free CM after 24 h of CPC monoculture and EV were isolated by differential centrifugation (Beckmann Coulter LE-80K Optima), as described before (26 (link)). In short, CM was centrifuged by subsequently 2,000 x g, 10,000 x g and 100,000 x g steps. The resulting 100,000 x g pellet was resuspended in PBS and centrifuged again at 100,000 x g. The washed EV pellet was resuspended in a small volume of PBS and stored at 4°C until further use.
Subsequently, we also isolated EV using size-exclusion chromatography (SEC), as described before (26 (link)). Serum-free CM was collected after 24 h of CPC monoculture and was centrifuged at 2,000 x g to remove debris, after which it was filtered (0.45 μm). CM was then concentrated to 5 mL using 100-kDa molecular weight cut-off (MWCO) Amicon Ultra Centrifugal Filters (Merck Millipore). The concentrated CM was subsequently loaded onto a S400 highprep column (GE Healthcare, Uppsala, Sweden) using an AKTAStart (GE Healthcare) containing a 280 nm flow cell. The fractions containing EV were pooled, concentrated using 100 kDa MWCO Amicon Ultra Centrifugal Filters, and filtered (0.45 μm). The EV protein concentration was determined with the microBCA protein assay kit (Thermo Scientific). EV were stored at 4°C and added to the medium of hfCF-laden GelMA in a concentration of 3 μg/mL.

All proteins used in this study were produced essentially as previously outlined, i.e. hHDM-FH (53 (link)). Briefly, for mHDM-FH, a single stable expressing clone was selected for protein production and scaled up in static tissue culture before transfer to roller bottles for 10 days (in the absence of Hygromycin B). Cell debris was removed by centrifugation and supernatant was sterile-filtered prior to loading on a 2 ml HiTrap NHS-activated high-performance column (GE Healthcare), which had been previously coupled (following manufacturers guidance) with the anti-mFH monoclonal antibody 2A5 (gift from Prof C. Harris, Newcastle UK), using an AKTA START (GE Healthcare) at a flowrate of 1 ml/min. Washes of 10 column volumes were performed prior to elution. Elution was achieved using 0.1M Glycine (pH of 2.7) and eluate was collected into 1M Tris pH 9.0. All protein-containing fractions were then combined and buffer exchanged into PBS using a PD-10 column (GE Healthcare). Proteins were further polished using a gel filtration column (Superdex 600, GE Healthcare) equilibrated with PBS buffer with an ÄKTA Purifier set to a flow rate of 0.5ml/min. One ml fractions were collected and stored at -80°C until needed.

Anti-S-IgGs and anti-RBD-IgGs were obtained using S-Sepharose and RBD-Sepharose on an Akta Start chromatograph (GE Life Sciences, New York, NY, USA) and as described in [46 (link)]. The sorbents with immobilized S-protein and RBD were prepared according to the standard protocol using CNBr Sepharose (GE Life Sciences, New York, NY, USA) and our previously published works [46 (link),63 (link)]. An equimolar mixture of 10 IgG preparations was applied to 3 mL of RBD-Sepharose pre-equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl (TBS). The fraction eluted with the acidic buffer was neutralized by adding 1/10 v/v 1.0 M Tris-HCl, pH 8.8, and then dialyzed against 20 mM Tris-HCl, pH 7.5. The IgG fraction not bound to RBD-Sepharose was applied to a 10 mL S-Sepharose column pre-equilibrated with TBS. The IgGs were eluted with a two-step gradient: 50 mM Tris-HCl, pH 7.5, containing 1.0 M NaCl and 0.1 M Gly-HCl, pH 2.6. The resulting fraction was also dialyzed against 20 mM Tris-HCl, pH 7.5.