Escherichia coli strain

Pichia pastoris (Invitrogen) strain GS115 as the expression host and pPICZαA (Invitrogen) as the expression vector were used for heterologous protein expression. Escherichia coli strain Top10 (Invitrogen) cells were used in DNA recombinant procedures. Buffered complex medium, containing glycerol (BMGY; Invitrogen) was used for growing the cells before induction and buffered complex medium containing methanol (BMMY; Invitrogen) was used as induction medium. Restriction enzymes were purchased from Fermentase. Anti-His antibody for detection of recombinant histidine-tagged urate oxidase was purchased from Roche Applied Science. Other reagents were obtained from standard commercial sources.

Human cDNA for PKM2 was cloned into pET28a⁺ with a N-terminal His tag and purified from Escherichia coli strain BL21 (Invitrogen) using Ni-Agarose beads (Qiagen) as described previously²⁵ .

Bacterial strains and plasmids used in this study were listed in Table 1. Escherichia coli strain DH5α and BL21 (DE3) and Saccharomyces cerevisiae used in this study were purchased from Invitrogen. The E. coli DH5α strain and E. coli BL21 (DE3) were used for plasmids preparation and for protein overexpression respectively. S. cerevisiae was used for gene for cloning. E. coli DH5α and E. coli BL21 (DE3) were cultured in Luria–Bertani (LB) broth in construction of strains and plasmids. S. cerevisiae was cultured in YPD medium. Antibiotics were added at final concentration of 50 μg/mL for kanamycin, 34 μg/mL for chloramphenicol and 100 μg/mL for ampicillin when necessary.