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  • Title: Synthetic leaders with potential BiP binding mediate high-yield secretion of correctly folded insulin precursors from Saccharomyces cerevisiae.
    Author: Kjeldsen T, Pettersson AF, Hach M, Diers I, Havelund S, Hansen PH, Andersen AS.
    Journal: Protein Expr Purif; 1997 Apr; 9(3):331-6. PubMed ID: 9126604.
    Abstract:
    Secretion leaders are essential for expression of many heterologous proteins including insulin in yeast. The function of secretion leaders and their interaction with the secretory pathway is not clear. To determine what constitutes functional pre-pro-leader sequences in Saccharomyces cerevisiae, synthetic leader sequences for secretion of the insulin precursor were developed by a combination of rational design and stepwise systematic optimization. The synthetic leaders efficiently facilitate secretion of the insulin precursor from S. cerevisiae when compared with the alpha-factor leader, leading to a high yield of correctly folded insulin precursor in the culture supernatant. The synthetic leaders feature two potential N-linked glycosylation sites which are efficiently glycosylated during secretion. Pulse-chase analysis indicates that the synthetic leaders/insulin precursor fusion protein have a prolonged residence in the endoplasmic reticulum compared to the alpha-factor leader/insulin precursor fusion protein. The longer transition time in the endoplasmic reticulum mediated by the synthetic leaders might provide additional time for correct folding of the insulin precursor and account for the increased fermentation yield.
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