Deae sepharose

DNA encoding the truncated human ACE2 ectodomain (residues 19–613) with a C-terminal AVI-tag and 6xHis-tag was synthesized (IDT) and subcloned into a pHLSec expression plasmid. Plasmid DNA was used for transient expression in Expi293F cells using ExpiFectamine 293 Transfection Kits (Thermo Fisher Scientific). Expression supernatant was harvested at day 6 and dialyzed into 10 mM Tris pH 8.0. ACE2 was subsequently purified using weak anion exchange (DEAE Sepharose, Cytiva), followed by size-exclusion chromatography (Superdex 200, Cytiva). Protein was then biotinylated enzymatically using BirA enzyme and further purified using strong anion exchange (MonoQ, Cytiva).

The preparation of hSTOM(SPFH) and its alanine-substituted mutants were performed as described previously (Tsuruta et al., 2012 (link)). The plasmid vectors carrying the R125A and K188A mutants of GST-tagged hSTOM(SPFH) were prepared according to a standard PCR mutagenesis method using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Briefly, the GST-tagged hSTOM(SPFH) (wild type and the mutants) was expressed in Escherichia coli BL21 (DE3) grown in M9 minimal medium containing [¹⁵N]-NH₄Cl or LB medium under isopropyl-β-_D-1-thiogalactopyranoside (IPTG) induction. The supernatant of the sonicated cells was purified using DEAE Sepharose (Cytiva, Marlborough, MA, USA) and Glutathione Sepharose 4 Fast Flow (Cytiva). After the cleavage of the GST-tag using thrombin (Wako) at 25 ​°C, the protein was further purified using size exclusion chromatography with a Superdex 75 HR 26/60 column (Cytiva) equilibrated with 50 ​mM Tris/HCl buffer (pH 7.5) containing 150 ​mM NaCl. The purified proteins were concentrated and dialyzed with 50 ​mM sodium phosphate buffer (pH 6.0) containing 100 ​mM NaCl or 50 ​mM bis-Tris/HCl (pH 6.0) containing 100 ​mM NaCl, depending on the experiment.

Ascidian (Halocynthia roretzi) tunics were purchased from a fresh market in Tongyeong, Korea. The tunics were washed with tap water and kept at −40 °C. Chondroitin AC lyase (EC 4.2.2.5) from Arthrobacter aurenses, chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris, monosaccharide standards, 1,9-dimethylmethylene blue, glucuronolactone and toluidine blue-O were obtained from Sigma-Aldrich (St. Louis, MO, USA); DEAE-Sepharose was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Human follicle dermal papilla (HFDP) cells (C-12071) were purchased from PromoCell GmbH (Heidelberg, Germany). Cells were cultured in follicle dermal papilla cell growth medium (C-26501, PromoCell GmbH, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), 50 units/mL of penicillin and 50 mg/mL of streptomycin. All the other reagents were of analytical grade.