Directed Evolution of Proteins Based on Mutational Scanning

Methods in Molecular Biology
Carlos G Acevedo-RochaManfred T Reetz

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 eng...Continue Reading

Citations

May 23, 2018·Applied Microbiology and Biotechnology·Aitao LiManfred T Reetz
Nov 20, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·Victor SayousCarlos G Acevedo-Rocha
Jan 13, 2021·ACS Synthetic Biology·Kaori HiragaDanny A Bitton
Aug 1, 2020·ACS Catalysis·Liliana Calzadiaz-RamirezCarlos G Acevedo-Rocha

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