A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli.

Autor:	Des Soye BJ; Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL 60208, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA; Center for Synthetic Biology, Northwestern University, Evanston, IL 60208, USA., Gerbasi VR; Proteomics Center of Excellence, Northwestern University, Evanston, IL 60208, USA., Thomas PM; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA; Proteomics Center of Excellence, Northwestern University, Evanston, IL 60208, USA; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA., Kelleher NL; Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL 60208, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA; Proteomics Center of Excellence, Northwestern University, Evanston, IL 60208, USA; Department of Chemistry, Northwestern University, Evanston, IL 60208, USA; Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA., Jewett MC; Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL 60208, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA; Center for Synthetic Biology, Northwestern University, Evanston, IL 60208, USA; Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA; Simpson Querrey Institute, Northwestern University, Chicago, IL 60611, USA. Electronic address: m-jewett@northwestern.edu.
Jazyk:	angličtina
Zdroj:	Cell chemical biology [Cell Chem Biol] 2019 Dec 19; Vol. 26 (12), pp. 1743-1754.e9. Date of Electronic Publication: 2019 Nov 06.
DOI:	10.1016/j.chembiol.2019.10.008
Abstrakt:	The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins via amber suppression provides access to novel protein properties, structures, and functions. Historically, poor protein expression yields resulting from release factor 1 (RF1) competition has limited this technology. To address this limitation, we develop a high-yield, one-pot cell-free platform for synthesizing proteins bearing ncAAs based on genomically recoded Escherichia coli lacking RF1. A key feature of this platform is the independence on the addition of purified T7 DNA-directed RNA polymerase (T7RNAP) to catalyze transcription. Extracts derived from our final strain demonstrate high productivity, synthesizing 2.67 ± 0.06 g/L superfolder GFP in batch mode without supplementation of purified T7RNAP. Using an optimized one-pot platform, we demonstrate multi-site incorporation of the ncAA p-acetyl-L-phenylalanine into an elastin-like polypeptide with high accuracy of incorporation and yield. Our work has implications for chemical and synthetic biology. (Copyright © 2019 Elsevier Ltd. All rights reserved.)
Databáze:	MEDLINE
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