It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.
A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.
Fermentation strategies for recombinant protein production in Pichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, application of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.
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