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).