3.1 Cloning and expression of rAGL in P. pastoris
The coding region derived from O. sativa AGL excluding the fragment of its original signal peptide but with an N -terminal 6-histidine tag, was synthesized and cloned into the plasmid pGAP9K, which was fused with α-Factor signal sequence of the plasmid for secretion in Pichia under the control of PGAP. Subsequently, the genomic integration was carried out by transformation of the linearized plasmid DNA that was digested using Psh AI, intoP. pastoris GS115. The transformed cells were spread on MD agar plates. Positive transformants containing high copy number of AGL gene were obtained through screening on YPD plates with different concentrations of geneticin. As a result, 12 clones were selected from the plates containing 4 mg/mL geneticin, among which four clones were confirmed by colony PCR with four sets of primers (Supplementary Figure 1) and selected for the subsequent expression experiments.
Expression levels of four selected clones were tested in the shake flask culture. Samples were collected every 12 h, and protein concentration and enzyme activity in the culture supernatant were detected. The growth profiles of cell mass (OD600) for the four recombinant clones were similar and could reach the highest value of 58.4±3.7 at 72 h, when the one having the maximum protein concentration (0.114 mg/mL) and enzyme activity (1.48 U/mL) was used for scale-up fermentation and rAGL production.