Pet 30a

The catalytic domain of PP5 (167-489 amino acids, PP5c) was ligated to the pMD-19T vector. The PaPP5c-pMD-19T plasmid was digested at the Nde1 and Xho1 site and then inserted into pET-30a (+) (TaKaRa) vector (digested at the same restriction sites) with T4 ligase (ThermoFisher). The recombinant plasmid was transformed into E. coli BL21 (DE3) cells. The positive transformant was cultured in LB liquid medium including 50 mg/mL of kanamycin at 37 °C with shaking at 200 rpm until the OD600 reached 0.5. We then added 0.1 mM of IPTG and 1mM of MnCl₂ to induce protein expression at 22 °C, with shaking at 200 rpm for 24 h. Recombinant cells were centrifuged at 4 °C, 8000 g, for collecting the cell pellet. The harvested cell pellet was resuspended in PBS (10 mM), lysed with lysozyme (1 mg/mL), and then disrupted with sonication on ice for 10 min. After sonication, the supernatant was subjected to protein purification. The supernatant was purified with a Ni-NTA column (Smart-Lifesciences, Changzhou, China) using gradient concentrations of imidazole buffer (50–250 mM) to dissolve the protein. The purified protein was detected by 15% SDS-PAGE. Dialysate (4 mM of MnCl₂, 10 mM of Tris-HCl, pH 8.0) was employed to dialyze the protein overnight at 4 °C. The concentration of purified protein was determined by the BCA method using the Easy II Protein Quantitative Kit (TransGene, Beijing, China).

MmedCSP3 was PCR-amplified using gene-specific primers (Table 1). The sample cDNA of the antennae was used as the template. The PCR product was first cloned into a pEASY-T3 Vector (Takara, Dalian, China) and then excised and cloned into an expression vector pET-30a (+) for expression in prokaryotic BL21 (DE3) cells (Takara, Dalian, China). The transformation of the strain with pET-30a (+) / MmedCSP3 was incubated in 500 mL of LB medium with 100 μg / ml kanamycin at 37°C. When the OD of the culture reached 0.4–0.6, the protein was induced with 1 mM isopropylthio-β-galactoside (IPTG) at 16°C and vibrated for 16 h at 200 rpm. The cultures were harvested by centrifugation at 12000 × g for 25 min at 4°C. The supernatant was obtained by sonication, purified by Ni ion affinity chromatography (ÄKTA avant 25, GE Healthcare, USA). Soon after the His-tag was removed with recombinant enterokinase (Novoprotein, Shanghai, China), followed by a second purification mentioned above. A 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was conducted to check the size and purity of recombinant MmedCSP3.