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Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

Morawski, Birgit and Lin, Zhanglin and Cirino, Pat and Joo, Hyun and Bandara, Geethani and Arnold, Frances H. (2000) Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris. Protein Engineering, 13 (5). pp. 377-384. ISSN 0269-2139. https://resolver.caltech.edu/CaltechAUTHORS:20111201-083504577

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Abstract

The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants. Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells. We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host. Here we examined expression of HRP in Saccharomyces cerevisiae. Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S.cerevisiae culture supernatant compared with wild-type, as measured on ABTS [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] (260 units/l/OD_(600)). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD_(600) in shake flasks. The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1093/protein/13.5.377DOIArticle
http://peds.oxfordjournals.org/content/13/5/377PublisherArticle
ORCID:
AuthorORCID
Cirino, Pat0000-0002-7245-6205
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2000 Oxford University Press. Received November 12, 1999. Accepted February 20, 2000. Revision received January 31, 2000. This research was supported by the Office of Naval Research and Maxygen Corporation. Pat Cirino is supported by a predoctoral fellowship from the National Science Foundation.
Funders:
Funding AgencyGrant Number
Office of Naval Research (ONR)UNSPECIFIED
Maxygen, Inc.UNSPECIFIED
NSF Predoctoral FellowshipUNSPECIFIED
Subject Keywords:directed evolution; horseradish peroxidase; random mutagenesis; recombination; yeast
Issue or Number:5
Record Number:CaltechAUTHORS:20111201-083504577
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20111201-083504577
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:28264
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:13 Apr 2012 18:06
Last Modified:03 Oct 2019 03:30

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