After methanol induction for 72 h, the transformant with highest xylanase activity showed the activity of 101.5 U mL−1 in the culture supernatant.
HSA fusion proteins were purified from media conditioned by transformed Pichia pastoris cells and induced with 0.5% vol/vol methanol, as previously described [ 18, 19, 28], except that the methanol induction phase of protein production was extended to 96 hours.
While there are several studies analyzing cellular reactions of P. pastoris to methanol induction in context of recombinant protein production [ 14– 18], the response of non-recombinant strains to the different carbon sources is largely unknown.
Elimination of the 6-hr fed-batch step, addition of a sorbitol pulse and sorbitol co-feed during methanol induction, and extension of the induction stage from approximately 72 h to approximately 96 h are the main changes introduced into the new revised fermentation process.
After 48 h of methanol induction, several clones were compared by means of an S-Tag assay.
After the ramp period, the methanol feed was maintained at 11 mL/L/hr and the 50% sorbitol feed was maintained at 3 mL/L/hr until the end of fermentation (after approximately 96 h of methanol induction).
Na-GST-1 protein product of 24 kDa was detectable after 22 h of methanol induction (Fig. 1B, lane 3) and accumulated in the culture supernatant throughout the length of the fermentation (Fig. 1B, lanes 4 6).
LHP7 was expressed in Pichia pastoris, the total concentration of secreted protein reached 0.906 g/L after 108 h of methanol induction.
