Functional expression of Coprinus cinereus peroxidase in Pichia pastoris

Su Jin Kim, Jeong Ah Lee, Keehoon Won, Yong Hwan Kim, Bong Keun Song

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

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).

Original languageEnglish
Pages (from-to)731-735
Number of pages5
JournalProcess Biochemistry
Volume44
Issue number7
DOIs
StatePublished - Jul 2009

Keywords

  • Coprinus cinereus peroxidase
  • Glycosylation
  • Heterologous protein expression
  • Pichia pastoris
  • Protein secretion

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