The candidate proteins were searched from nonredundant and expressed sequence tag (EST) against the NCBI database (http://blast.ncbi.nlm.nih.gov/ ) and gene/protein database of E. granulosus (http://www.gendb.org/Homepage/ ). Each primer containing BamHI and HindIII restriction sites was prepared (Supplementary Table S1 ). The genes were amplified from the E. granulosus cDNA library by PCR: 35 cycles for 5 min at 94°C, 45 sec at 94°C, 1 min at 58°C, 45 sec at 72°C, 1 min at 72°C, with final extension for 10 min at 72°C [19 (link)]. The amplicons were purified by QIAquick Gel extraction kit (Qiagen, Hilden, Germany). The plasmids were ligated to pET-28a or pGEX-6p-1 vector and transformed into Escherichia coli DH5α (Takara, Shiga, Japan).
Recombinant plasmids were transformed into E. coli BL21 (DE3) and cultured in the Luria-Bertani medium supplemented with antibiotics (50 μg/ml). Recombinant proteins were induced with 1 mM isopropyl-β-D-thiogalactopyranoside for 4 hr at 37°C. The cells were lysed and centrifuged at 20,000 g for 1 hr. Recombinant proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose chromatography (Qiagen) or Glutathione Sepharose 4B affinity column (GE healthcare).
Recombinant plasmids were transformed into E. coli BL21 (DE3) and cultured in the Luria-Bertani medium supplemented with antibiotics (50 μg/ml). Recombinant proteins were induced with 1 mM isopropyl-β-D-thiogalactopyranoside for 4 hr at 37°C. The cells were lysed and centrifuged at 20,000 g for 1 hr. Recombinant proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose chromatography (Qiagen) or Glutathione Sepharose 4B affinity column (GE healthcare).