Eudiplozoon nipponicum

Roudnický, Pavel, Vorel, Jiří, Ilgová, Jana, Benovics, Michal, Norek, Adam, Jedličková, Lucie, Mikeš, Libor, Potěšil, David, Zdráhal, Zbyněk, Dvořák, Jan, Gelnar, Milan & Kašný, Martin, 2018, Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea), Parasite (Paris, France) 25 (61), pp. 1-13 : 3

Expression of recombinant E. nipponicum serpin (rEnSerp1) and its purification

Overnight culture (5 mL, OD = 2) was inoculated into 500 mL of a fresh LB medium (L3022, Sigma-Aldrich) with ampicillin (100 µg/mL). Bacteria were grown at a temperature of 30 °C at 200 rpm until OD = 0.8 was reached, then cooled down to 12 °C and induced by IPTG (0.5 mM) for 18 h. Harvested cells were lysed in a binding buffer (specifications below) by sonication on ice, and subsequently centrifuged at 10 000 · g for 40 min at 4 °C. Soluble rEnSerp1 was purified from the supernatant by immobilised metal affinity chromatography ( IMAC) using Ni 2+ HisTrap columns (1 mL, GE Healthcare) connected to FPLC ( ÄKTA, GE Healthcare).

Buffers used in the individual stages of purification were composed as follows: binding buffer – 50 mM Tris-HCl pH 8, 10 mM imidazole, 300 mM NaCl, 5% glycerol, 0.05% Tween; elution buffer – 50 mM Tris-HCl pH 8, 500 mM imidazole, 300 mM NaCl, 5% glycerol, 0.05% Tween. Protein purity was evaluated in a 4–15% TGX SDS-PAGE gel under reducing conditions (Bio-Rad Mini-Protean). In a sample buffer, wells contained 5 µg of protein. Gel documentation was performed in a calibrated densitometer GS-900 TM (Bio-Rad). Recombinant EnSerp1 was deprived of imidazole and salts using PD-10 columns (GE Healthcare), and then transferred into a 100 mM HEPES buffer (pH 7.5 with 300 mM NaCl, 10 mM CaCl 2, and 0.05% Brij L23), i.e. the same buffer that was used for inhibition assays. Protein concentration was determined using a Quaint-iT TM Protein Assay Kit (Life Technologies) and a SpectraMax i3 fluorometer (Molecular Devices).