E coli bl21 de3 plyss cells

Recombinant plasmids of pfsra were transformed into E. coli BL21 (DE3) pLysS cells (TransGen Biotech, Beijing, China) and then grown in Luria Bertani broth containing ampicillin (50 µg/mL) at 37 °C for 12 h. The culture was induced by 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; TransGen Biotech, Beijing, China) when the optical density at 600 nm (OD₆₀₀) reached 0.4–0.6, and was allowed to grow for another 8 h at 37 °C. Finally, the culture was harvested by centrifugation at 4000× g for 30 min, and the protein was purified by TianLin Biotech (Wuxi, China).

The nucleotide sequence encoding the full-length of pysra was obtained from the PlasmoDB website (PYYM_1014900), synthesized by TianLin Biotech (Wuxi) with codon optimization for expression in E. coli system, and cloned into the pET30a vector. The pysra-F1, pysra-F2, and pysra-F3 fragments were amplified by polymerase chain reaction (PCR) from the full-length gene, and the PCR products were purified by 2% agarose gel electrophoresis and cloned into a pET32a vector (Primer sequences in Table S1). Recombinant plasmids of pysra were transformed into E. coli BL21 (DE3) pLysS cells (TransGen Biotech) and then grown in Luria Bertani broth containing ampicillin (50 µg/mL) at 37°C for 12 h. The culture was induced by 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; TransGen Biotech) when the optical density at 600 nm (OD600) reached 0.4–0.6 and was allowed to grow for another 8 h at 37°C. Finally, the protein was purified by TianLin Biotech (Wuxi).