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Int. J. Mol. Sci. 2015, 16(10), 23127-23142; doi:10.3390/ijms161023127

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

1
Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a, 1060 Vienna, Austria
2
Department of Chemistry, University of Natural Resources and Life Sciences, 1180 Vienna, Austria
*
Author to whom correspondence should be addressed.
Academic Editor: Qiang “Shawn” Chen
Received: 12 August 2015 / Revised: 14 September 2015 / Accepted: 15 September 2015 / Published: 24 September 2015
(This article belongs to the Special Issue Protein Engineering)
View Full-Text   |   Download PDF [1030 KB, uploaded 24 September 2015]   |  

Abstract

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins. View Full-Text
Keywords: horseradish peroxidase; glyco-engineering; strain engineering; bioreactor cultivation; OCH1; site-directed mutagenesis horseradish peroxidase; glyco-engineering; strain engineering; bioreactor cultivation; OCH1; site-directed mutagenesis
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Capone, S.; Ćorajević, L.; Bonifert, G.; Murth, P.; Maresch, D.; Altmann, F.; Herwig, C.; Spadiut, O. Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris. Int. J. Mol. Sci. 2015, 16, 23127-23142.

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