P pastoris expression manual

P. pastoris, GS115 and Escherichia coli, DH5α strains were purchased from Invitrogen (Carlsbad, CA, USA). The pHBM905BDM plasmid was constructed in our laboratory. All culture media, including minimal dextrose (MD,1.34% YNB, 2% dextrose, 4 × 10⁻⁵% biotin), buffered minimal glycerol (BMGY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10⁻⁵% biotin, 1% glycerol), and buffered minimal methanol (BMMY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10⁻⁵% biotin, 0.5% methanol) were prepared, as described in the P. pastoris expression manual (Invitrogen).

Pichia pastoris GS115 (his4⁻), pGAPZa and pPIC9K used for the protein expression were obtained from Invitrogen (Thermo Fisher Scientific). Escherichia coli TOP10 or DH5α strain was used as the host for recombinant DNA construction work. E. coli was grown in Luria–Bertani (LB) medium at 37 °C with 100 μg/mL ampicillin or 50 μg/mL zeocin where necessary. Buffered minimal glycerol (BMGY) medium, buffered minimal methanol (BMMY) medium and minimal dextrose (MD) medium were prepared following the P. pastoris expression manual (Invitrogen). Mouse anti-His monoclonal antibody and mouse anti-Flag monoclonal antibody were purchased from Genscript Bio-Technologies (Nanjing, China). Con A-Biotin was purchased from Vector Laboratories. HRP-conjugated secondary antibody and HRP- conjugated Streptavidin was purchased from ZSGB-Bio (Beijing, China). All other chemicals and solvents were bought from Sangon-Biotech (Shanghai, China).

Pichia pastoris GS115 and E. coli DH5α/BL21 strains were purchased from Invitrogen (Shanghai, China). The pHBM905BDM plasmid was constructed in our laboratory. All culture media, including Luria–Bertani (LB), minimal dextrose (MD), buffered minimal glycerol (BMGY) and buffered minimal methanol (BMMY) were prepared as described in the P. pastoris expression manual (Invitrogen).