Hen egg white lysozyme

E. coli cultures producing Tle3 or its periplasmic-targeted fusion were grown to an OD₆₀₀ of 0.6 before the induction of construct expression with 100 µM IPTG. Three hours post-induction, cells were harvested by centrifugation at 8000 g at 4 °C. Pellets were normalised to an OD₆₀₀ of 20, resuspended in 200 µl spheroplast buffer (200 mM Tris-HCl pH 8.0, 500 µM EDTA and 500 mM sucrose) containing 50 µg hen egg-white lysozyme (Sigma) and incubated on ice for 15 min. Then, 720 µl spheroplast buffer was diluted 1 : 1 with distilled water and added to the spheroplasts. The spheroplasts were separated from the periplasmic fraction by centrifugation at 5000 g for 10 min at 4 °C and the isolated periplasmic fraction was centrifuged once more to remove residual spheroplasts. The periplasmic fraction was adjusted to the equivalent of an OD₆₀₀ of 10 in Laemmli buffer.

Hen egg white lysozyme, lysostaphin, nisin, mutanolysin, and melittin were purchased from Sigma Aldrich. The following bacteria were used for antimicrobial activity assays and RNA isolation: Staphylococcus aureus ATCC 6538 (American Type Culture Collection, Manassas, VA, USA), Bacillus cereus ATCC 10876, Enterococcus faecalis ATCC 29212, Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC 27957, Streptococcus equi subsp. zooepidemicus ATCC 43079, Streptococcus iniae KCTC 3657 (Korean Collection for Type Cultures, Daejeon, Korea), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Salmonella typhimurium ATCC 14028. E. faecalis and streptococci were cultured in brain heart infusion broth (BD Bioscience) because of their slow growth in LB medium. All other bacteria were cultured in LB medium.

Immune parameters in plasma were assessed in 8 fish per tank (N = 16). Plasma lysozyme activity was evaluated by turbidimetric assay, according to Ellis^{31 (link)}, based on the addition of the samples to a standard bacterial suspension of Micrococcus lysodeikicus. The absorbance decrease caused by bacterial lysis was measured by readings at 0.5 min and 4.5 min after addition. Values were standardized using hen egg white lysozyme (Sigma, Portugal). Plasma peroxidase levels were determined by the chemical reduction of 3,3_, 5,5_—tetramethyl benzidine hydrochloride (Sigma, Portugal), according to Quade and Roth^{32 (link)}.

Methyl α-d-glucoside (98%), vinyl methacrylate (>98.0%), thioflavin T (ThT, 98%), (methyl sulfoxide)-d₆ (DMSO-d₆, 99.9 atom% D) and deuterium oxide (D₂O, 99.9 atom% D) were obtained from J&K Chemicals (Beijing, China). Novozym 435 was purchased from Novozymes Biotech (Tianjin, China). Hen egg white lysozyme (HEWL, ≥90%) was received from Sigma-Aldrich St. Louis (Shanghai, China). Azodiisobutyronitrile (AIBN, 99%) was purchased from Sigma-Aldrich (Shanghai, China) and recrystallized before use. Distilled deionized water was prepared from a Millpore Filtration System (Millpore, Bedford, MA, USA). A high-precision and ready-to-use dialysis bag (Nominal 1000) was purchased from Shanghai Green Bird Science and Technology Development Co., Ltd. (Shanghai, China). The diperiodatocuprate(III) (Cu(III)) [15 (link)] and methyl 6-O-methacryloyl-α-d-glucoside (6-O-MMAGlc) [16 (link)] were synthesized according to the methods reported in the literature.

The chemical structures of the molecules used in this study are given in Figure 1. Trehalose (>99%), maltose monohydrate (>99%), and β-gentiobiose (>85%) were purchased from Sigma-Aldrich, lucifer yellow ethylenediamine (94%) was purchased from Setareh Biotech, and all were used without any further purification. Solutions for fluorescence experiments were prepared from ultrapure 18 MΩ water at concentrations of 200 μM for lucifer yellow ethylenediamine (LYen). All the measurements were taken at room temperature (~21 °C). Staphylococcal nuclease (SNase) was expressed and purified in our lab with the method described by Shortle and Meeker [31 (link)] and modifications mentioned by Byrne et al. [32 (link)] The protein concentration was determined to be 10.2 mg/mL using the extinction coefficient ε280 = 0.93 mg/mL·cm² [33 ]. The protein samples were stored at −80 °C in water. Prior to performing melting experiment, the samples were thawed and adjusted to final concentration of 1mg/mL. Hen egg white lysozyme (>90%) was purchased from Sigma-Aldrich, and samples for circular dichroism were prepared according to the protocol described by Greenfield et al. [34 (link)]. Samples were prepared in a 10 mM phosphate buffer, pH 7.2, and stored at 4 °C for up to one week prior to use. The final concentration of lysozyme samples was 0.83 mg/mL.

Assays were carried out in triplicate, using flat-bottomed 96-well untreated ELISA plates (Greiner). OBodies suspended in PBS pH 7.4 were first diluted 1∶2 serially over 11 steps, starting with a final assay concentration of 50 µM. A 25 µL aliquot of each OBody dilution was either added to 25 µL hen egg-white lysozyme (Sigma) at 648 nM (162 nM final concentration in assay) or 25 µL PBS pH 7.4 and allowed to equilibrate at room temperature for 15 min. To each OBody dilution, a 50 µL aliquot of substrate solution containing inactivated Micrococcus lysodeikticus cells (Sigma, M3770) suspended in PBS pH 7.4 was added to a final concentration of 0.4 mg/mL and plates were incubated at room temperature for 45 min before reading the absorbance at 450 nm in a BMG Fluostar Optima plate reader. The data were normalised and processed using Prism v5.04 (Graphpad) software and non-linear regression performed using the following model [Y = Bottom+(Top-Bottom)/(1+10<$>\vskip -3 \raster(60%)="rg1"<$>((LogIC₅₀−X)*HillSlope))] to obtain IC₅₀ values.

The following materials were used in this study: Hen egg-white lysozyme (Sigma-Aldrich, Steinheim, Germany), ultrapure trehalose (Cargill, Krefeld, Germany), ultrapure betaine (Fluka Biochemika, Buchs, Switzerland), firoin (Biotop, Berlin-Brandenburg, Germany), ultrapure ectoine and hydroxyectoine (Biomol, Hamburg, Germany), citric acid (Riedel-de Haen, Seelze, Germany), sodium hydroxide, dimethylsulfoxide (DMSO), Nile red and M. lysodeickticus (Sigma-Aldrich, Steinheim, Germany), phosphate buffered saline (PBS buffer 66 mM phosphate, pH 6.2) (Fluka Analytical, Steinheim, Germany), SYPRO orange protein gel stain, (Invitrogen, Eugene, Oregon, USA),