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Functional expression of Coprinus cinereus peroxidase in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
35131 45077 2009 5 PDF Available
Title
Functional expression of Coprinus cinereus peroxidase in Pichia pastoris
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).

Keywords
YPDS, yeast extract peptone dextrose sorbitol; BMMY, buffered minimal methanol complex; BMGY, buffered minimal glycerol yeast extract medium; SEC, size exclusion chromatography; BCA, bicinchoninic acid; BSA, bovine serum albumin; PBS, phosphate buffer sol
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Functional expression of Coprinus cinereus peroxidase in Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 44, Issue 7, July 2009, Pages 731–735
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us