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.