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Functional Expression of Horseradish Peroxidase in E. coli by Directed Evolution

Lin, Zhanglin and Thorsen, Todd and Arnold, Frances H. (1999) Functional Expression of Horseradish Peroxidase in E. coli by Directed Evolution. Biotechnology Progress, 15 (3). pp. 467-471. ISSN 8756-7938. https://resolver.caltech.edu/CaltechAUTHORS:20180823-144310796

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Abstract

In an effort to develop a bacterial expression system for horseradish peroxidase (HRP), we inserted the gene encoding HRP into the pET‐22b(+) vector (Novagen) as a fusion to the signal peptide PelB. A similar construct for cytochrome c peroxidase (CcP) leads to high CcP activity in the supernatant. Expression of the wild‐type HRP gene in the presence of isopropyl‐β‐d‐thiogalactopyranoside (IPTG) yielded no detectable activity against ABTS (azinobis(ethylbenzthiazoline sulfonate) ). However, weak peroxidase activity was detected in the supernatant in the absence of IPTG. The HRP gene was subjected to directed evolution: random mutagenesis and gene recombination followed by screening in a 96‐well microplate format. From 12 000 clones screened in the first generation, one was found that showed 14‐fold higher HRP activity than wild‐type, amounting to ∼110 μg of HRP/L, which is similar to that reported from laborious in vitro refolding. No further improvement was obtained in subsequent generations of directed evolution. This level of expression has nonetheless enabled us to carry out further directed evolution to render the enzyme more thermostable and more resistant toward inactivation by H_2O_2. These results show that directed evolution can identify mutations that assist proteins to fold more efficiently in Escherichia coli. This approach will greatly facilitate efforts to “fine‐tune” those many enzymes that are promising industrial biocatalysts, but for which suitable bacterial or yeast expression systems are currently lacking.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/bp990037rDOIArticle
https://doi.org/10.1021/bp990292bDOIErratum
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1999 American Institute of Chemical Engineers (AIChE). Accepted March 10, 1999. Dr. L. Giver is gratefully acknowledged for help with the initial PCR cloning of cytochrome c peroxidase. This work was supported by the Office of Naval Research (N0014-96-1-340).
Errata:Subsequent to the publication of this article, sequencing errors were uncovered by the authors, who report that mutant 1A6 in fact has no mutations in the gene encoding HRP. As a result, they retract the article in its entirety.
Funders:
Funding AgencyGrant Number
Office of Naval Research (ONR)N0014-96-1-340
Issue or Number:3
Record Number:CaltechAUTHORS:20180823-144310796
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180823-144310796
Official Citation:Lin, Z. , Thorsen, T. and Arnold, F. H. (1999), Functional Expression of Horseradish Peroxidase in E.coli by Directed Evolution. Biotechnol Progress, 15: 467-471. doi:10.1021/bp990037r
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89102
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:23 Aug 2018 22:29
Last Modified:03 Oct 2019 20:13

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