Ceramic hydroxyapatite

Human sCD38 (a gift from H.C. Lee) was obtained as previously reported with some modification [17 (link)]. The sCD38 transformed P. pastoris strains were grown at 28°C for 24 hours in 400 mL of buffered glycerol-complex medium (BMGY) and induced with methanol. When the activity of NGD or ε-NAD was maximal, culture media were harvested by centrifugation at 8,000 X g for 20 minutes. The sCD38 in the media was precipitated by 70% ammonium sulfate fractionation at 4°C and the precipitate collected by centrifugation at 14,000 X g for 20 minutes at 4°C. The precipitate was resuspended and dialyzed with 15 mM Tris-HCl pH 7.4. The sample was loaded onto Reactive RED 120-agarose (Sigma-Aldrich), and sCD38 was separated using a linear salt gradient. Fractions showing ADP-ribosyl cyclase activity were pooled, applied to ceramic hydroxyapatite (Bio-Rad), and eluted using a linear phosphate gradient. Thereafter, purified sCD38 was separated with HiLoad 26/600 Superdex 75 (GE Healthcare). Purified sCD38 was loaded onto a High Capacity Endotoxin Removal Spin Column (Pierce) and eluted samples were aliquoted and stored at –70°C until use.

Prophenoloxidase was purified from the hemolymph of day-3 5th instar H. armigera larvae according to the protocol reported described (30 (link)). Briefly, 10 ml hemolymph was collected from larval body and pooled into ice-cold saturated ammonium sulfate (AS). AS saturation (35-50%) was collected and loaded on column prepacked with Ceramic Hydroxyapatite (Bio-Rad, Hercules, CA, United States). The fractions with cetylpyridinium chloride (CPC) activated PO activity were combined and applied through Concanavalin A Sepharose column (Sigma-Aldrich, St. Louis, MO, United States). The flow-through fraction was applied to a Phenyl Sepharose 6 Fast Flow (low sub) column (GE Healthcare, Little Chalfont, United Kingdom). Fractions containing PO activity were applied to a Superdex 200 column (ÄKTApurifier; GE Healthcare). Purified PPO were stored at −80°C before analysis.