Improved production of recombinant ovine interferon-tau by mut(+) strain of Pichia pastoris using an optimized methanol feed profile

Recombinant ovine interferon-tau (r-oIFN-tau) production by Pichia pastoris was studied using methanol as the sole carbon source during induction. The cells were grown on glycerol up to a certain cell density before induction of the AOX1 promoter by methanol for expression of the recombinant protein. Cell growth on methanol has been modeled using a substrate-feed equation, which served as the basis for an effective computer control of the process. The r-oIFN-tau concentration in the culture began to decline despite continued cell growth after 50 (+/- 6) h of induction, which was associated with an increase in proteolytic activity of the fermentation broth. A specific growth rate of 0.025 h(-1) was found to be optimal for r-oIFN-tau production. No significant improvement in r-oIFN-tau production was observed when the specific growth rate was stepped up before the critical point when r-oIFN-tau concentration started decreasing during fermentation. However, best results were obtained when the specific growth rate was stepped down from 0.025 to 0.02 h(-1) at 38 h of induction, whereby the active production period was prolonged until 70 h of induction and the broth protease activity was correspondingly reduced. The corresponding maximum protein yield was 391.7 mg x L(-1) after 70 h of fermentation. The proteolytic activity could be reduced by performing fermentations at specific growth rates of 0.025 h(-1) or below. The recombinant protein production can be performed at an optimal yield by directly controlling the methanol feed rate by a computer-controlled model. The production profile of r-oIFN-tau was found to be significantly different from other secreted and intracellular recombinant protein processes, which is an indication that recombinant protein production in Pichia pastoris needs to be optimized as individual processes following established principles.