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Functional expression ofCoprinus cinereusperoxidase inPichia pastoris

Authors
Journal
Process Biochemistry
1359-5113
Publisher
Elsevier
Publication Date
Volume
44
Issue
7
Identifiers
DOI: 10.1016/j.procbio.2009.03.004
Keywords
  • Coprinus Cinereusperoxidase
  • Heterologous Protein Expression
  • Protein Secretion
  • Glycosylation
  • Pichia Pastoris

Abstract

Abstract A Coprinus cinereus peroxidase (CiP) was successfully expressed by the methylotrophic yeast Pichia pastoris. The 1095-bp gene encoding peroxidase from C. cinereus was cloned with a highly inducible alcohol oxidase ( AOX1) promoter and integrated into the genome of P. pastoris. The recombinant CiP (rCiP) fused with the α-mating factor pre-pro leader sequence derived from Saccharomyces cerevisiae accumulated neither inside the cell nor within the wall, and were efficiently secreted into the culture medium. SDS-PAGE and immunoblot analysis revealed that the rCiP was not hyper-glycosylated and its α-factor signal sequence was correctly processed. It was also found that the kinetic properties of rCiP were similar to those of native CiP. In order to produce large amounts of rCiP, the high cell density cultivation of recombinant P. pastoris was carried out in a fermentor with fed-batch mode. The peroxidase activity obtained in a 5 l fermentor cultivation became about 6 times (1200 U/ml) higher than that in shake-flask cultures (200 U/ml).

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