Lysozyme from chicken egg

mAb-A as well as the FcγRIIIa purified proteins were kindly provided by Roche Diagnostics (Penzberg, Germany). Both FcγRIIIa purified proteins consisted of the extracellular domain, linked to an AviTag and a LALA-PG mutated IgG1 Fc domain (17 (link)). 7.5 M ammonium acetate (AmAc) solution, lysozyme from chicken egg, glacial acetic acid and hydrogen chloride were purchased from Sigma-Aldrich (Steinheim, Germany). Deionized water was obtained using a Milli-Q purification system from EMD Millipore (Burlington, MA). 10 and 30 kDa Vivaspin MWCO filters were purchased from Satorius (Göttingen, Germany).

The coding sequence of Vd2LysM was amplified from V. dahliae cDNA and cloned into the pETSUMO (Invitrogen) expression vector according to the manufacturer's instructions prior to E. coli Origami (DE3) transformation. A single transformant was selected and grown in Luria broth medium until an optical density at 600 nm (OD₆₀₀) of 0.9 was reached. Heterologous production of Vd2LysM was induced with 1 mm Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 26°C during ∼20 h. Cell pellets were lysed using lysozyme from chicken egg (Sigma, St Louis, MO, USA) and Vd2LysM was purified from the soluble protein fraction using an Ni²⁺‐NTA Superflow column (Qiagen). Purified protein was dialysed against 200 mm NaCl and concentrated over Amicon ultracentrifugal filter units (Molecular weight cut‐off (MWCO) = 3 kDa; Millipore, Billerica, MA, USA).

Protein absorption and release study was carried out as previously reported [9 (link)]. Model proteins lysozyme from chicken egg (Sigma-Aldrich, UK) and bovine serum albumin (BSA, Sigma-Aldrich, UK) were solubilized in HBSS (Thermo-Fisher, UK) at 10 µg/mL and 100 µg/mL, respectively. To investigate the effect of the nanoclay particles on drug release, 3D scaffolds were printed using nanocomposite (LAB) and Laponite-free controls (alginate-bone-ECM (AB)) to allow the absorption of the compounds of interest after ionic crosslinking. The 3D-printed constructs (n=3) were soaked in lysozyme or BSA for 1 h, and their release was monitored over 24 h. BSA and lysozyme were quantified with a RAPID kit (Sigma-Aldrich, UK) using a GloMax Discover microplate reader (Promega). The supernatant was collected after 1, 2, 4, 8, 10, 20, and 24 h following adsorption. Collagenase D (from Clostridium histolyticum, Roche Diagnostics GmbH) was added 24 h after adsorption to stimulate material degradation and cargo release. BSA and lysozyme release was quantified after 1, 2, 4, 8, 10, 20, and 24 h.