2.1 Strains, media, and chemicals
Escherichia coli DH5α (TransGen Biotech, Beijing, China) andP. pastoris GS115 (Invitrogen, Carlsbad, USA) were used as the host strains for DNA cloning and protein expression, respectively. The plasmid pPIC9K (Invitrogen, Carlsbad, USA) was selected as a backbone vector to generate the recombinant expression vectors for P. pastoris.
E. coli DH5α was grown at 37°C in Luria-Bertani (LB) medium (50 mg/mL Kanamycin supplemented when necessary). Yeast strains were cultivated in the yeast extract peptone dextrose (YPD) medium, buffered minimal glycerol (BMGY) medium, or minimal dextrose (MD) medium following the pPIC9K manual. YPD medium containing different concentrations of G418 sulfate (geneticin, Yuanye Bio-Technology, Shanghai, China), which is applied to select geneticin resistant strains.
FastPure Plasmid Mini Kit and Taq DNA polymerase were obtained from Vazyme (Nanjing, China). The restriction enzymes were purchased from New England Biolabs (Beverly, USA). The nucleotide sequences used in this study were synthesized by Genscript (Nanjing, China). D-Glucose assay kit based on the combined action of glucose oxidase (GOD) and peroxidase (POD), was obtained from Yuanye Bio-Technology Co., Ltd (Shanghai, China). AA-2G was purchased from Tokyo Chemical Industry Co. Ltd. (Japan).