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.