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The effect of process conditions on productivity and glycosylation of cystatin C in P. pastoris and DNA microarray analysis of amino acid limitations in S. cerevisiae culture Pritchett, Jason

Abstract

Human cystatin C is a cysteine-proteinase inhibitor with several potential therapeutic applications. A recombinant variant of cystatin C with two potential sites for N-linked glycosylation (Nakamura, Ogawa et al. 2000) was selected for expression in the yeast Pichia pastoris. Glycosylation has been shown to significantly improve the heat stability and activity of recombinant cystatin C (Nakamura, Ogawa et al. 1998). When using standard fermentation protocols however, most cystatin C is not glycosylated. Thus, the effects of induction pH, temperature, and nitrogen sources on cystatin C productivity and glycosylation were examined in 250 mL shake flasks and 2-litre bioreactors. The pH and temperature were studied over the ranges of 5.2 - 6.8, and 21 - 35°C respectively. Nitrogen sources examined include ammonium hydroxide, peptone and amino acid supplements. Nitrogen source was the most significant parameter. The maximum cystatin C productivity and glycosylation was obtained under conditions of 20 g•L⁻¹ peptone, 20 g•L⁻¹ amino acid mix, and 0 g•L⁻¹ ammonium hydroxide resulting in 13.6 nmol-gDCW⁻¹•h⁻¹ cystatin C with 30% glycosylated species, a five-fold increase from standard fermentation conditions. In a separate study using S. cerevisiae, DNA microarray analysis was used to monitor gene expression changes resulting from amino acid limitations in fermentation media. Experiments were performed to examine leucine and glutamine limitations imposed on batch cultures. Recent literature results from Gasch (Gasch, Spellman et al. 2000) and Natarajan (Natarajan, Meyer et al. 2001) were also re-analyzed. The results show that both leucine and glutamine biosynthesis genes were differentially expressed as much as 13-fold under complete amino acid starvation and genes involved in leucine and histidine biosynthesis were significantly up-regulated under mild leucine limitation and histidine starvation with 3AT (3-aminotriazol). Additional amino acid starvation experiments revealed that leucine starvation resulted in a 2-fold up-regulation of the leucine biosynthesis genes LEU1, LEU2 and LEU4. However, changes in gene expression for glutamate biosynthesis genes under glutamic acid starvation resulted in no distinguishable trends.

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