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  Directed Evolution of Proteins Based on Mutational Scanning

Acevedo-Rocha, C. G., Ferla, M., & Reetz, M. T. (2018). Directed Evolution of Proteins Based on Mutational Scanning. In U. T. Bornscheuer, & M. Höhne (Eds.), Methods in Molecular Biology (pp. 87-128). New York: Springer. doi:10.1007/978-1-4939-7366-8_6.

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 Creators:
Acevedo-Rocha, Carlos G.1, 2, 3, Author              
Ferla, M.4, Author
Reetz, Manfred T.1, 2, Author              
Affiliations:
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
2Philipps-Universität Marburg, Fachbereich Chemie, ou_persistent22              
3Biosyntia ApS, 2100, Copenhagen, Denmark, ou_persistent22              
4Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK, ou_persistent22              

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Free keywords: Synthetic biology. Directed evolution. Protein engineering. Site-directed mutagenesis. Saturation mutagenesis. Scanning mutagenesis. Mutability landscapes. Deep mutational scanning. Cytochrome P450 monooxygenase. Stereoselectivity.
 Abstract: Directed evolution has emerged as one of the most effective protein engineering methods in basic research as well as in applications in synthetic organic chemistry and biotechnology. The successful engineering of protein activity, allostery, binding affinity, expression, folding, fluorescence, solubility, substrate scope, selectivity (enantio-, stereo-, and regioselectivity), and/or stability (temperature, organic solvents, pH) is just limited by the throughput of the genetic selection, display, or screening system that is available for a given protein. Sometimes it is possible to analyze millions of protein variants from combinatorial libraries per day. In other cases, however, only a few hundred variants can be screened in a single day, and thus the creation of smaller yet smarter libraries is needed. Different strategies have been developed to create these libraries. One approach is to perform mutational scanning or to construct “mutability landscapes” in order to understand sequence–function relationships that can guide the actual directed evolution process. Herein we provide a protocol for economically constructing scanning mutagenesis libraries using a cytochrome P450 enzyme in a high-throughput manner. The goal is to engineer activity, regioselectivity, and stereoselectivity in the oxidative hydroxylation of a steroid, a challenging reaction in synthetic organic chemistry. Libraries based on mutability landscapes can be used to engineer any fitness trait of interest. The protocol is also useful for constructing gene libraries for deep mutational scanning experiments.

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Language(s): eng - English
 Dates: 2017-10-312018-02-16
 Publication Status: Published in print
 Pages: 42
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1007/978-1-4939-7366-8_6
 Degree: -

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Title: Methods in Molecular Biology
  Subtitle : Protein Engineering
Source Genre: Series
 Creator(s):
Bornscheuer, Uwe T.1, Editor
Höhne, Matthias2, Editor
Affiliations:
1 Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Greifswald, Germany, ou_persistent22            
2 Protein Biochemistry, Institute of Biochemistry, Greifswald University, Greifswald, Germany, ou_persistent22            
Publ. Info: New York : Springer
Pages: - Volume / Issue: 1685 Sequence Number: - Start / End Page: 87 - 128 Identifier: DOI: 10.1007/978-1-4939-7366-8_6
ISBN: 978-1-4939-7364-4