Isopropyl β d thiogalactoside

Aflatoxin B₁ (AFB₁), zearalenone (ZEN), deoxynivalenol (DON), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,6-dimethoxy phenol (DMP), syringaldazine (SGZ), and methyl syringate were purchased from Sigma-Aldrich (St. Louis, MO, USA). FB₁ (fumonisin B₁) and OTA (ocharatoxin A) were purchased from Pribolab (Beijing, China). DNA polymerase, T4 ligase, acetonitrile, and trifluoroacetic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Vanillin, p-coumaric, syringic acid, syringaldehyde, caffeic acid, 1-hydroxybenzotriazole (HBT), gallic acid, isopropyl-β-D-thiogalactoside (IPTG), and kanamycin were purchased from Solarbio (Beijing, China). Ni-NTA agarose was purchased from QIAGEN (Hilden, Germany). The fungal laccase from Ganoderma sp. was purchased from Sunson (Yinchuan, Ningxia, China). Plant extracts from E. brevicornu, C. sativus L., L. angustifolia, A. officinalis, and S. tenuifolia were purchased from Ciyuan Biotech (Xi’an, Shanxi, China). All other chemicals were of analytical grade or chromatographically pure, and were commercially available.

Among Giardia-secreted proteins highly expressed in the trophozoite culture supernatant as described before [31 (link)], PNPO (GL50803_5810), PPIB (GL50803_17163), and tenascin-1/2/3 (GL50803_114815/10330/16833) were randomly chosen and expressed in a prokaryotic expression system. The genes encoding these proteins were amplified using the primer sets shown in Table S2, and then cloned into the pCold I vector (TaKaRa, Ohtsu, Japan). The resulting plasmids pCold I-PNPO/PPIB/tenascins were transformed into E. coli strain BL21 (DE3) cells. Protein expression was induced with 1 mM isopropyl β-d-thiogalactoside (Solarbio, Beijing, China) at 16 °C for 20 h. The harvested cells were lysed using an ultrasonic cell crusher (XO-650D; Xianou, Nanjing, China). Precipitated proteins and supernatants were subjected to SDS-PAGE analysis. Before treatment, the recombinant proteins were purified with a HisTrap HP nickel column (SMART, Changzhou, China). We then used an endotoxin ELISA kit (Meimian Biotech, Yancheng, China) to measure if the purified proteins were contaminated with endotoxins that may be responsible for the observed activation. If contaminated, proteins were further purified with Endotoxin Removal Beads (Smart-Lifesciences Biotechnology, Changzhou, China).

All genes, bacterial strains, and plasmids used in this study are listed in Table 1. All CaFLS mutants used in this study are listed in Table 2. The gene and protein sequences used in this study are listed in Supplementary materials S1, S2, respectively. All the wild-type genes and mutanted genes were synthesized and sequenced by Sangon Biotech (Shanghai, China). The restriction nuclease was obtained from Sangon Biotech (Shanghai, China). Hesperetin and diosmetin was procured from Macklin (Shanghai, China) and Sigma-Aldrich (St. Louis, MO, United States). diosmetin, 4′-O-methyl taxifolin, and tamarixetin reference material were purchased from Sigma-Aldrich. Isopropyl-β-D-thiogalactoside (IPTG), Luria-Bertani broth (LB), and kanamycin were purchased from Solarbio (Beijing, China).