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Purification of a recombinant plant peroxidase produced in Pichia pastoris by a simple 2-step strategy.

Authors :
Spadiut O
Rossetti L
Dietzsch C
Herwig C
Source :
Protein expression and purification [Protein Expr Purif] 2012 Dec; Vol. 86 (2), pp. 89-97. Date of Electronic Publication: 2012 Sep 28.
Publication Year :
2012

Abstract

The enzyme horseradish peroxidase (HRP), which is frequently applied in industry and medicine, is primarily isolated from plant. This purification procedure is costly and the obtainable amount of HRP from the horseradish root is low. However, recombinant HRP (rHRP) produced in yeast is hyperglycosylated rendering the subsequent purification cumbersome and the recombinant production of HRP in yeast not competitive. In this study, we screened different common techniques to develop a fast and efficient purification strategy for hyperglycosylated rHRP expressed in Pichia pastoris. We demonstrated that the extensive glycosylation pattern on the surface of rHRP masked its physico-chemical properties, which is why standard purification strategies were rather unsuccessful. Only switching the strategies to a flowthrough mode gave satisfactory results. After determining the optimal operation conditions in a multivariate Design of Experiments approach, we present a simple 2-step strategy for the purification of hyperglycosylated rHRP. Combining a hydrophobic charge induction chromatography operated in flowthrough mode and a size-exclusion chromatography, we were able to purify rHRP more than 12-fold from a specific activity of 80U/mg to more than 1000U/mg.<br /> (Copyright © 2012 Elsevier Inc. All rights reserved.)

Details

Language :
English
ISSN :
1096-0279
Volume :
86
Issue :
2
Database :
MEDLINE
Journal :
Protein expression and purification
Publication Type :
Academic Journal
Accession number :
23026679
Full Text :
https://doi.org/10.1016/j.pep.2012.09.008