Histrap ff ni sepharose columns

pPICZα vector containing XEG1-His^{34 (link)} or empty vector was transformed into Pichia pastoris strains X33 (Muts⁺) or KM71 (Muts). P. pastoris was cultured overnight in the YPD medium at 30 °C and subsequently grown in the BMGY (Buffered Glycerol-Complex Medium) and BMMY (Buffered Methanol-Complex Medium) (pH = 6.5) for protein expression. The recombinant protein XEG1-His or EV was purified from the supernatant of the P. pastoris culture harboring pPICZα-XEG1-His or pPICZα using the AKTA™ avant 25 (GE Healthcare) through the HisTrap™ FF Ni Sepharose Columns (5 ML, 17525501; GE Healthcare) and HiTrap™ Desalting Prepacked Columns (5 ML, 29048684; GE Healthcare).

The full-length ORF of BCG1 was amplified using the genomic cDNA of PH-1, and then cloned into the pPICZα vector^{63 (link)}. The resulting pPICZα::BCG1 construct was transformed into Pichia pastoris X-33 (Muts⁺), which was cultured overnight in the YEPD (yeast extract-peptone-dextrose) medium at 30 °C. For protein expression, P. pastoris transformants were grown in the BMGY (Buffered Glycerol Complex Medium) for 24 h and then transferred to BMMY (Buffered Methanol-Complex Medium) (pH = 6.5). The recombinant protein BCG1-His was purified from the culture supernatant of P. pastoris using HisTrap FF Ni Sepharose Columns (GE Healthcare). The expression and purification of 6 × His tagged BCG1^E118A, BCG1^E209A, BCG1^ΔGQ, XYLA, XYLB, AfBCG1, and MoBCG1 recombinant proteins were similarly performed as described above.