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

Benjamin J. Des Soye; Vincent R. Gerbasi; Paul M. Thomas; Neil L. Kelleher; Michael C. Jewett

doi:10.1016/j.chembiol.2019.10.008

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

Benjamin J. Des Soye, Vincent R. Gerbasi, Paul M. Thomas, Neil L. Kelleher, Michael C. Jewett^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

59 Scopus citations

Abstract

Des Soye et al. create and optimize a strain of Escherichia coli that expresses T7 RNA polymerase so that lysates prepared from the strain are enriched with sufficient polymerase to catalyze high-yielding cell-free transcription and translation reactions. Using the resulting platform, the authors synthesize products containing up to 40 non-canonical amino acids.

Original language	English (US)
Pages (from-to)	1743-1754.e9
Journal	Cell Chemical Biology
Volume	26
Issue number	12
DOIs	https://doi.org/10.1016/j.chembiol.2019.10.008
State	Published - Dec 19 2019

Funding

This work was supported by the Army Research Office W911NF-18-1-0200 and W911NF-18-1-0181 , National Science Foundation (NSF) grant MCB-1716766 , the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the David and Lucille Packard Foundation , and the Camille Dreyfus Teacher-Scholar Program (to M.C.J.). This research was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under grant P41 GM108569 from the National Institute of General Medical Sciences , National Institutes of Health. B.J.D. is a recipient of the NSF Graduate Research Fellowship and was supported in part by NIH Predoctoral Biotechnology training grant T32GM008449 . We thank Prof. Brad Bundy for providing pY71 plasmids and Prof. Peter G. Schultz for providing the pEVOL-pAcF plasmid. Research reported in this publication was made possible in part by the services of the NUSeq Core Facility, which is supported by the Northwestern University Center for Genetic Medicine, Feinberg School of Medicine, and Shared and Core Facilities of the University's Office for Research. The US Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the US Government. This work was supported by the Army Research Office W911NF-18-1-0200 and W911NF-18-1-0181, National Science Foundation (NSF) grant MCB-1716766, the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the David and Lucille Packard Foundation, and the Camille Dreyfus Teacher-Scholar Program (to M.C.J.). This research was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health. B.J.D. is a recipient of the NSF Graduate Research Fellowship and was supported in part by NIH Predoctoral Biotechnology training grant T32GM008449. We thank Prof. Brad Bundy for providing pY71 plasmids and Prof. Peter G. Schultz for providing the pEVOL-pAcF plasmid. Research reported in this publication was made possible in part by the services of the NUSeq Core Facility, which is supported by the Northwestern University Center for Genetic Medicine, Feinberg School of Medicine, and Shared and Core Facilities of the University's Office for Research. The US Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the US Government. B.J.D. designed research, performed all genomic engineering and CFPS reactions, performed top-down mass spectrometry of ELP-40 constructs, analyzed all data, and wrote the paper. V.R.G. performed top-down mass spectrometry of all sfGFP constructs as well as ELP-20 and ELP-30 constructs. P.M.T. and N.L.K. supervised and advised on all top-down mass spectrometry. M.C.J. directed research, analyzed data, and wrote the paper. The authors have filed a patent application related to this work with US Serial No. 15/651484.

Keywords

cell-free protein synthesis
chemical biology
genetic code expansion
genome engineering
in vitro transcription and translation
non-canonical amino acids
synthetic biology

ASJC Scopus subject areas

Biochemistry
Molecular Medicine
Molecular Biology
Pharmacology
Drug Discovery
Clinical Biochemistry

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10.1016/j.chembiol.2019.10.008

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

abstract = "Des Soye et al. create and optimize a strain of Escherichia coli that expresses T7 RNA polymerase so that lysates prepared from the strain are enriched with sufficient polymerase to catalyze high-yielding cell-free transcription and translation reactions. Using the resulting platform, the authors synthesize products containing up to 40 non-canonical amino acids.",

keywords = "cell-free protein synthesis, chemical biology, genetic code expansion, genome engineering, in vitro transcription and translation, non-canonical amino acids, synthetic biology",

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note = "Funding Information: This work was supported by the Army Research Office W911NF-18-1-0200 and W911NF-18-1-0181 , National Science Foundation (NSF) grant MCB-1716766 , the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the David and Lucille Packard Foundation , and the Camille Dreyfus Teacher-Scholar Program (to M.C.J.). This research was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under grant P41 GM108569 from the National Institute of General Medical Sciences , National Institutes of Health. B.J.D. is a recipient of the NSF Graduate Research Fellowship and was supported in part by NIH Predoctoral Biotechnology training grant T32GM008449 . We thank Prof. Brad Bundy for providing pY71 plasmids and Prof. Peter G. Schultz for providing the pEVOL-pAcF plasmid. Research reported in this publication was made possible in part by the services of the NUSeq Core Facility, which is supported by the Northwestern University Center for Genetic Medicine, Feinberg School of Medicine, and Shared and Core Facilities of the University's Office for Research. The US Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the US Government. Funding Information: This work was supported by the Army Research Office W911NF-18-1-0200 and W911NF-18-1-0181, National Science Foundation (NSF) grant MCB-1716766, the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the David and Lucille Packard Foundation, and the Camille Dreyfus Teacher-Scholar Program (to M.C.J.). This research was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health. B.J.D. is a recipient of the NSF Graduate Research Fellowship and was supported in part by NIH Predoctoral Biotechnology training grant T32GM008449. We thank Prof. Brad Bundy for providing pY71 plasmids and Prof. Peter G. Schultz for providing the pEVOL-pAcF plasmid. Research reported in this publication was made possible in part by the services of the NUSeq Core Facility, which is supported by the Northwestern University Center for Genetic Medicine, Feinberg School of Medicine, and Shared and Core Facilities of the University's Office for Research. The US Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the US Government. B.J.D. designed research, performed all genomic engineering and CFPS reactions, performed top-down mass spectrometry of ELP-40 constructs, analyzed all data, and wrote the paper. V.R.G. performed top-down mass spectrometry of all sfGFP constructs as well as ELP-20 and ELP-30 constructs. P.M.T. and N.L.K. supervised and advised on all top-down mass spectrometry. M.C.J. directed research, analyzed data, and wrote the paper. The authors have filed a patent application related to this work with US Serial No. 15/651484. Publisher Copyright: {\textcopyright} 2019 Elsevier Ltd",

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

Abstract

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Keywords

ASJC Scopus subject areas

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