Ni nta

Restriction enzymes, T4 DNA ligase, DL5000 DNA markers, and DNA loading buffer were from Takara, plasmid extraction kits from Tiangen Biotech Co. Ltd. (Beijing, China), and gel extraction kits were bought from Axygen Biosciences (Union City, CA, USA. Protein markers from Cell Signaling Technology (Danvers, MA, USA), Trans 2xnEasy Taq PCR Super Mix, 4S Green Plus Nucleic acid stain, protein loading buffer from Yeasen Biotechnology Co. Ltd. (Shanghai, China), SDS gel stain wash buffer from Beyotime Biotechnology Co. Ltd. (Shanghai, China), R250 from Ourchem, Sinopharm Chemical Reagent Co. Ltd. (shanghai, China), Ni-NTA from TransGen Biotech Co. Ltd., (Shanghai, China). pET28a from Novagen (Foster City, CA, USA), PMD18T, Takara Co. Ltd. (Dalian, China).

The ECEA production strain was cultured in 150 mL of SGGP medium for 72 h. The fermentation broth was then centrifuged at 4000× g 4 °C for 30 min. The supernatant was passed through a 0.45 μm NY filter (Shimadzu, Shanghai, China). The filtrate was loaded to a Ni-NTA (TransGen Biotech, Beijing, China) equipped with an ÄKTA start protein purifying system (GE, Boston, MA, USA). The elution buffer used for ECEA purification was 100 mM Tris-HCl (pH 8.0), containing 300 mM imidazole. The chromatographic peaks corresponding to ECEA were collected and concentrated using an ultrafilter tube (Sigma-Aldrich, Shanghai, China). The chromatographic fractions and crude protein samples (cell pellets) were analyzed using an SDS-PAGE gel and Coomassie staining (Figure S7).