Bacterial strains and plasmids used in this study are listed in Supporting Information, Table S1. Photorhabdus luminescens TT01 was grown in Luria–Bertani (LB) medium at 28 °C, and strains of E. coli were grown in LB medium at 37 °C. Escherichia coli DH5α was used as the host Ipilimumab for recombinant DNA cloning. Escherichia coli BL21 (DE3) was used as the host for expression of binary toxin genes. Plasmid pET28a (Novagen) was used as expression vector in BL21 (DE3). Plasmid pETDuet-1 (Novagen) was used as co-expression vector in BL21 (DE3). Total DNA was

extracted from P. luminescens TT01 using the alkali lysis method. It was used as template for amplification of plu1961 and plu1962 (GenBank accession no. BX571865). Oligos used in this study were listed in Table S2. Oligo pair Plu1961-F/Plu1961-R was used to amplify plu1961, and Plu1962-F/Plu1962-R was used to amplify plu1962. Both PCR products were double-digested by EcoRI/SalI and cloned into pET28a to generate plasmids pET-plu1961 and pET-plu1962, respectively. For co-expression of Plu1961 and Plu1962 in BL21 (DE3), Co1961-F/Co1961-R and Co1962-F/Co1962-R were used to amplify plu1961 and plu1962, respectively. PCR products of plu1961 and plu1962 were double-digested

by PstI/SalI and NdeI/XhoI, respectively, and cloned into pETDuet-1 sequentially to generate co-expression plasmid pET-pluBi. All the plasmids were confirmed by DNA sequencing. Plasmids Selleckchem Ganetespib pET-plu1961, pET-plu1962, and pET-pluBi were transformed into BL21 (DE3), and resultant strains were designated as BL21 (plu1961), BL21 (plu1962), and BL (Bi),

respectively. Recombinant strains were grown in LB medium with kanamycin (50 μg mL−1) or ampicillin (100 μg mL−1) at 37 °C to an OD600 between 0.6 and 0.8. Then, isopropyl-beta-d-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mmol L−1. After IPTG induction for 4 h, (-)-p-Bromotetramisole Oxalate aliquots of 1 mL bacteria culture were sampled and harvested by centrifugation (10 000 g, 1 min). Pellets were washed three times with distilled water and suspended in 0.1 mL lysis buffer (50 mmol L−1 NaH2PO4, 300 mmol L−1 NaCl, 10 mmol L−1 imidazole, pH 8.0). Then, cells were lysed by sonication and centrifuged at 10 000 g for 2 min. The supernatant was collected, and 10 μL aliquots were taken for SDS-PAGE. Soluble binary toxins (Plu1961 and Plu1962) were directly purified on 1-mL HisTrapTM HP prepacked columns (GE Healthcare), using an AKTA Purifier system (GE Healthcare; flow rate 1 mL min−1). The column was equilibrated in His A buffer (20 mmol L−1 sodium phosphate, 0.5 mol L−1 NaCl, 20 mmol L−1 imidazole, pH 7.4). Proteins were eluted using a step gradient up to 0.5 mol L−1 imidazole in His A buffer. Fractions were analyzed by SDS-PAGE, and the protein content of the pools was determined using the Bio-Rad Bradford reagent. The purified proteins were dialyzed against PBS buffer prior to application.