Gel filtration standard

To analysis the monomeric or polymeric state of Igs in trout nasal mucus, gel filtration analyses were performed using as described previously for gut [16 (link)] and gill mucus [15 (link)]. In short, fractions containing the IgM, IgT or IgD were separated by gel filtration using a Superdex-200 FPLC column (GE Healthcare). The column was previously equilibrated with cold PBS (pH 7.2), and protein fractions were eluted at 0.5 ml min ^-1 with PBS using a fast protein LC instrument with purifier systems (GE Healthcare). Identification of IgM, IgD and IgT in the eluted fractions was performed by western blot analysis using anti-IgM, anti-IgD and anti-IgT antibodies, respectively. A standard curve was generated by plotting the elution volume of the standard proteins in a Gel Filtration Standard (Bio-Rad) against their known molecular weight, which was then used to determine the molecular weight of the eluted IgT, IgM and IgD by their elution volume.

Release of haeme from wild-type NdCld and variants was tested by incubation of the respective proteins with horse heart apo-myoglobin (Sigma) [5 ,34 ]. Apo-myoglobin was prepared using a modified extraction method by Teale [35 ] as described previously [5 ]. Thirty micromolars of NdCld samples were incubated with 40 μM apo-myoglobin in 50 mM phosphate buffer, pH 7.0, for 1 h at room temperature. Size-exclusion chromatography was performed to separate NdCld and myoglobin. HPLC (Shimadzu prominence LC20) was equipped with a refractive index detector (RID-10A, Shimadzu), a diode array detector (SPD-M20A, Shimadzu) and a MALLS-detector (Multi-Angle-Laser-Light-Scattering; WYATT Heleos Dawn8+ plus QELS; software ASTRA 6). The column (Superdex 200 10/300 GL, GE Healthcare) with particle size of 13 μm was equilibrated with the running buffer (Dulbecco PBS, 200 mM NaCl). The experiments were performed at a flow rate of 0.75 ml·min⁻¹, 80 μl of the protein solution (containing 30 μM NdCld and 40 μM apo-myoglobin) were injected. Untreated NdCld samples (wild-type, K141E, E210A, R173Q/E210A), holo- and apo-myoglobin as well as Bio-Rad Gel Filtration Standard (#151-1901) containing also holo-myoglobin from horse heart, were injected as references. Haeme transfer was observed by extracting elution profiles at 280 and 409 nm from the diode array detector.

The PdMCD WT and variants (Q115A, T116A, and E117A) were initially purified as follows. Cells were lysed by ultrasonification, and the lysate was cleared by ultracentrifugation at 50,000 rcf for 45 min at 4 °C and subsequently filtered through a 0.45-μm filter. The cleared lysate was loaded onto a 1-ml HisTrap FF column (GE Healthcare), and unbound protein was removed with 20 ml of 20 mm Tris-HCl, pH 7.9, 500 mm NaCl, 75 mm imidazole. The protein was eluted in 20 mm Tris-HCl, pH 7.9, 500 mm NaCl, and 500 mm imidazole and subsequently desalted with a HiTrap 5-ml desalting (GE Healthcare) column in 20 mm Tris-HCl, pH 7.9, 200 mm NaCl. After the addition of 20% glycerol, the enzymes were concentrated to 10 mg ml⁻¹ and incubated with 500 μm FAD for 1 h on ice. The enzymes were diluted to 2 mg ml⁻¹ in 20 mm Tris-HCl, pH 7.9, 200 mm NaCl. 100 μl were injected onto a Superdex 200 Increase 10/300 GL size-exclusion column (GE Healthcare) with a flow rate of 0.7 ml min⁻¹ and a running buffer of 20 mm Tris-HCl, pH 7.9, 200 mm NaCl. The size-exclusion calibration curve was obtained using the gel filtration standard (Bio-Rad).

For SEC experiments, 20 μg of Pry1CAP was injected onto a Yarra SEC-2000 column (Phenomenex, Torrance, CA) at flow-rate of 0.5 ml/min with a Shimadzu Prominence series HPLC (Kyoto, Japan) using PBS pH 7.4 as the mobile phase. The elution was monitored with a photo diode array detector (Shimadzu). The system was calibrated using Bio-Rad gel filtration standard (Hercules, CA) consisting of proteins with molecular weights of 670, 158, 44, 17 and 1.35 kDa. Data analysis was performed on the 280 nm wavelength extracted chromatograph using Shimadzu LCsolution version 1.25. Experiments were conducted in triplicate.