Recombinant Vector Expression and Purification in Pichia pastoris

The recombinant vectors of Anman and mutants were linearized by restriction endonuclease Sal al restriction endonuclease. The linear vector was transferred into P. pastoris X33 and incubated in yeast extract peptone dextrose (YPD) solid medium containing 100 mg/mL bleomycin (zeocin) at 30°C for 72 h. Colonies were picked and incubated in 30 Ml YPD liquid medium at 30°C and 220 rpm for 24 h, and cells were then collected by centrifugation at 8,000 rpm and transferred to 30 mL YP medium containing 0.5% methanol at 28°C and 220 rpm for 72 h. Methanol was added every 12 h to maintain its concentration at 0.5% for induction of enzyme production.
The induced fermentation broth was centrifuged at 4°C at 8,000 rpm, and the collected supernatant was dialyzed for 16 h in 0.1 M NaH₂PO₄-Na₂HPO₄ containing 20 mM NaCl (pH 5.5, buffer A) to remove salt and metal ions. Anion exchange chromatography was used for the purification of crude enzymes. Briefly, after pre-equilibrating the anion exchange column (Hi Trap™ 1 mL Q HP) with Buffer A, the dialyzed crude enzyme was loaded onto the column, followed by a linear gradient elution using Buffer A with Buffer B (Buffer A with 1 M NaCl), and the collection containing the enzyme activity was utilized for the subsequent study. The purification of the samples was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970 (link)). The Bradford method was used to determine the protein concentration (Bradford et al., 1976 (link)).