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Expression of human cystatin c by the methylotrophic yeast pichia pastoris Files, David Andrew


Human cystatin C is a cysteine-proteinase inhibitor with great potential as a therapeutic protein. The methylotrophic yeast, Pichia pastoris, was selected as a host to express a human cystatin C variant in which site directed mutagenesis was used to introduce two consensus sequences for N-linked glycosylation. Under the regulation of the methanol-inducible AOX1 gene promoter, both glycosylated and nonglycosylated cystatin were expressed simultaneously. Shake-flask experiments were performed to investigate the effects on cell density of glycerol and glucose as carbon sources and yeast extract, yeast nitrogen base, and ammonium sulfate as nitrogen sources. Glycerol was found to be superior to glucose, and the addition of yeast extract or ammonium sulfate (up to 4.3 gI-1) or yeast nitrogen base (up to 6.8 gI-1) had positive effects on the cell densities. The effects of pH and of feeding both methanol and glycerol during induction were investigated in a 2-liter bioreactor. The maximum expression was achieved at pH 6 and when only methanol was fed during induction. Glycerol feeding during induction showed potential to increase the concentration and yield of biologically active cystatin. The maximum concentration of biologically active cystatin in the culture supernatant was 54 μmolesI-1, equivalent to 0.72 gI-1 of human cystatin C. The maximum yield of biologically active cystatin was 1.0 μmoleg-1 dry cell weight. The effects of gene copy number as well as methanol utilization were considered. Resistance to the Zeocin™ antibiotic was used as a base for isolating transformants that were suspected to have a range of copy numbers. An inverse relationship between Zeocin™ resistance and active cystatin expression was observed. It is hypothesized that transformants with a higher resistance to Zeocin™ encountered inefficiencies in their secretion pathways. In terms of methanol utilization phenotype, the slow-growing transformant (Muts) expressed higher concentrations and higher yields of active cystatin. The fast-growing transformant (Mut+) experienced oxygen mass transfer limitations within the 2-liter bioreactor. [Scientific formulae used in this abstract could not be reproduced.]

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