Octyl sepharose 4 fast flow column

Paragonimus westermani adult worm extracts (PwAWE) were prepared as described previously [20 (link)]. In brief, Intact adult P. westermani worms kept in -80°C deep freezer were thawed and homogenized with Teflon-pestle homogenizer in phosphate buffered saline (PBS) containing protease inhibitor cocktail (1 tablet per 25 ml PBS). The extracts were centrifuged at 20,000 g at 4°C for 1 h and supernatants were collected. The supernatants were used as PwAWE. A 743 bp-long coding region of PwYF (AAG17056.1) was amplified by polymerase chain reaction with gene-specific primers 5’-AGCTCATATGGCCCCGGCAAGTGTTGACTG-3’ and 5’-GAAGTCTCGAGTTAGTGAATGATGGCG G-3’, incorporating NdeI and XhoI sites (underlined sequences). The recombinant plasmid was inserted into the pET28a(+) Escherichia coli expression vector. The expression fidelity was confirmed by DNA sequencing. rPwYF was induced by 0.5 mM isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 4 h. rPwYF purified by His-bind metal chelation resin (Novagen, Madison, WIS, USA) was examined by 15% reducing SDS-PAGE. Bacterial lipids were removed using Octyl-Sepharose 4 Fast Flow column (GE Healthcare, UK). The proteins were stored at -80°C.

The nucleotide sequences of CsGSTo1 (ANK78262) and 2 (ANK78263) were amplified from an adult C. sinensis cDNA library. The sequences were verified by sequencing, cloned into pET-28a(+) vector (Novagen, Madison, WI, USA) and transformed into Escherichia coli BL21 (DE3) (Thermo Fisher Scientific, Waltham, MA, USA). Bacterial cells were cultured with Luria–Bertani medium supplemented with kanamycin (50 µg/mL). Recombinant proteins, induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside, were purified by a nickel–nitrilotriacetic acid column (Qiagen, Hilden, Germany), followed by thrombin cleavage. The proteins were delipidated using an Octyl-Sepharose 4 Fast Flow column (GE Healthcare, Little Chalfont, UK) and bacterial endotoxin was removed using the Endotoxin Removal System (GenScript, Piscataway, NJ, USA). The homogeneity of the recombinant proteins was monitored by 15% reducing SDS-PAGE. Enzyme activity was assayed using dehydroascorbate substrate [13 (link)].