Recombinant enterokinase

The recombinant vectors were transformed into BL21 (ED3) competent cells. The positive clones were confirmed by sequencing. Protein expression was induced at 28 °C for 5 h with 0.5 mM isopropyl-β-D-thiogalactopryranoside (IPTG) when the culture OD₆₀₀ reached 0.6. The bacterial cells were harvested by centrifugation (8000 g, 10 min). The cellular pellets were resuspended in the lysis buffer (50 mM Tris-HCI, pH 8.0, 50 mM NaCI, 0.5% TritonX-100, 2 mg/mLIysozyme) and then sonicated (10 s, 5 passes). After centrifugation, the recombinant proteins were present as inclusion bodies. The inclusion bodies were solubilized and refolded following by the previously reported protocols^{54 (link),55 (link)}. Purification of the protein was accomplished by a Ni-NTA His·bind Resin column (7 sea Pharmatech Co., Shanghai, China), according to the manufacturer’s protocol. The His-tag was removed by digestion with recombinant enterokinase (Novoprotein, Shanghai, China), and the target proteins were purified again by the column mentioned above and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the purified proteins was measured using BCA protein assay kit (Beyotime, Shanghai, 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.