2.2 Plasmid construction and transformation
According to the P. pastoris GS115 chromosome, the sequence of PGAP (Supplementary Data 1) was synthesized and cloned between Sac I and Bam HI restriction sites of pPIC9K, generating the plasmid pGAP9K with PGAP. The AGL (UniProtKB accession no. Q653V7) encoding sequence from the Oryza sativa subsp. japonica (Rice) was synthesized with optimized codons for heterologous expression in P. pastoris (Supplementary Data 1), and inserted into the Eco RI site of pGAP9K with a 6-histidine tag at the N -terminus to replace the original signal peptide of AGL (1 - 33 amino acid residues), thus obtaining the plasmid pPIC9K-AGL. (Fig. 1).
Figure 1
Plasmid transformations were performed according to a condensed electroporation protocol previously described[40]. The plasmid pGAP9K-AGL was linearized withPsh AI and introduced into P. pastoris GS115 by electroporation to facilitate integration into the host genome. Multi-copy inserts of the transformants were selected by MD plates and the grown clones were transferred to YPD plates containing different concentrations of geneticin (0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, and 4 mg/mL) [41]. The geneticin-resistant clones were confirmed by Polymerase Chain Reaction (PCR) amplification with pairs of primers listed in Supplementary Table 1, and the PCR-positive clones were selected for the consequent expression tests. As a negative control, transformation was also performed using only the Psh AI linearized pGAP9K vector.