*3.2. Cloning, Expression and Purification of ScCDA2*

The *cda*<sup>2</sup> gene from *Saccharomyces cerevisiae* S288c (GenBank: NP\_013411.1) was amplified with upstream primer *Sc*CDA2-F:(5 -CATGCCATGGGAAGCTAATAGGGAAGATTTA-3 ) and downstream primer: *Sc*CDA2-R (5 -CCGCTCGAGGGACAAGAATTCTTTTATGTAATC-3 ). The target gene was digested and then ligated into a pPICZαA expression vector containing the N-terminal hexa histidine fusion tag coding region.

The *cda2* gene was recombined into a *Pichia pastoris* X-33 chromosome. Then the recombinant *Pichia pastoris* X-33 was induced by 0.5% methanol for 4 days, and methanol was added in batches every 24 h. The culture supernatant was collected by centrifugation at 8000× *g*, 4 ◦C for 20 min. The crude enzyme from the supernatant was concentrated using a 10 kDa ultrafiltration membrane. Then, the concentrated supernatant was purified by Ni-NTA Sepharose excel column (GE Healthcare). The pre-equilibrated buffer was subjected to Ni-NTA with buffer containing 20 mM PBS, pH 7.4, 300 mM NaCl and then washed with 50 mM PBS, pH 7.4, 300 mM NaCl, 20 mM imidazole. Finally, the target protein, eluted with 20 mM PBS, pH 7.4, 300 mM NaCl, 250 mM imidazole was obtained. Protein concentration was determined by using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).
