Lysozyme from chicken egg white

Cystamine dihydrochloride (CYS), 1.1 Carbonyldiimidazole (CDI), sodium alginate (Alginate, MW determined by viscosimetry was 140 kDa), dextran sulfate sodium salt (Dextran, MW 40 kDa), carboxymethyl-dextran sodium salt (CM-Dextran-MW 70 kDa), hyaluronic acid (low MW HA, 20–70 kDa), Rhodamine B isothiocyanate (RITC), DL-Dithiothreitol 99% (DTT), bovine serum albumin, 96% (BSA), dimethyl sulfoxide (DMSO), and lysozyme from chicken egg white, 90% were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Proteases used were Chymase (Sigma C8118), Cathepsin D (Sigma SRP6415), Cathepsin G (RP-77525), Cathepsin E (Biovision 7842), Pepsin (Roche 10108057001), Trypsin porcine pancreas (Sigma T0303-1G), Lysozyme from chicken egg white (Sigma L6876), Napsin A (RND 8489-NA). Digestion experiments were carried out with purified human hemoglobin (Sigma H7379) and recombinant or purified proteases. 100 μg hemoglobin (ca. 1.56 nmol) were digested with Chymase (in Tris-HCl 0.05 M, pH 8.0/0.26 M NaCl), Cathepsin D, G and E (in 0.2 M citrate buffer, pH 5.0), Pepsin (in 20 mM sodium acetate buffer, pH 3.5), Trypsin (in 0.1 M Tris–HCl, with 10 mM CaCl2, pH 8), Lysozyme (in 10 mM Tris-HCl, pH 8) or Napsin A (in 0.2 M NaCl, 0.1 M sodium acetate, pH 3.6). All proteases were used at a 1:100 molar ratio (15 pmol) and reactions were incubated at 37°C for 2 h.

Sodium chloride, sodium hydroxide, sodium
phosphate monobasic of p.a. quality, and o-phosphoric
acid (85%, HPLC) were purchased from Merck (Darmstadt, Germany). Hydrochloric
acid and Tris–HCl of p.a. quality were purchased from Honeywell
(Morris Plains, NJ, USA) and Kemika (Zagreb, Croatia), respectively.
The deionized water was purified with a Milli-Q purification system
from Millipore (Bedford, MA, USA) before use.
β-lactoglobulin
of ≥90% purity, lysozyme from chicken egg white of ≥98%
purity, and bovine serum IgG of ≥95% purity, all in the form
of lyophilized powder, were purchased from Sigma-Aldrich.

GI from Streptomyces rubiginosus was purchased from Hampton Research (HR7-098, Aliso Viejo, CA, USA), and was supplied in crystalline form and directly used for the SX experiment without post-crystallization, as previously reported [24 (link)]. The size and density of GI were < 60 × 60 × 40 μm³ and 3 × 10⁷ crystals/mL, respectively. Lysozyme from chicken egg white was purchased from Sigma-Aldrich (L6876, St. Louis, MO, USA) and crystallized as previously reported [40 (link)]. The size and density of lysozyme were < 60 × 60 × 40 μm³ and 3 × 10⁷ crystals/mL, respectively.

Extraction of total RNA from cells after induced overnight expression was done using the RNeasy Kit with optimized protocol for extraction from E. coli adapted from RNAprotect® Bacteria Reagent Handbook (Qiagen)⁶⁹ . Specifically, cell lysis was performed enzymatically using lysozyme from chicken egg white (Sigma) and proteinase K (NEB), followed by standard RNeasy protocol with on-column DNase I treatment (Roche) and final elution in RNase-free water.
RNA extraction from the purified VLP samples was performed based on the previously published protocol of extraction from Potyvirus particles^{70 (link)}. In brief, the sample was incubated in the presence of 1% SDS (w/v) at 55 °C for 5 min, followed by phenol-chloroform extraction. The extracted RNA was then precipitated by the addition of 0.5 initial sample volume of 7.5 M ammonium acetate and 2.5 volumes of cold absolute ethanol at −20 °C for 1 h, followed by 25 min centrifugation at 12,000 × g. After washing the pellet with 70% ethanol (v/v) and air-drying for 15–30 min at room temperature, the precipitated RNA was resuspended in DEPC-treated water and treated with Turbo DNase rigorous protocol (Thermo Fisher) to remove any potential DNA contaminants, followed by isolation with RNA Clean & Concentrator-5 kit (Zymo Research) and storage at −80 °C.