Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids

Rey W. Martin; Benjamin J. Des Soye; Yong Chan Kwon; Jennifer Kay; Roderick G. Davis; Paul M. Thomas; Natalia I. Majewska; Cindy X. Chen; Ryan D. Marcum; Mary Grace Weiss; Ashleigh E. Stoddart; Miriam Amiram; Arnaz K. Ranji Charna; Jaymin R. Patel; Farren J. Isaacs; Neil L. Kelleher; Seok Hoon Hong; Michael C. Jewett

doi:10.1038/s41467-018-03469-5

Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids

Rey W. Martin, Benjamin J. Des Soye, Yong Chan Kwon, Jennifer Kay, Roderick G. Davis, Paul M. Thomas, Natalia I. Majewska, Cindy X. Chen, Ryan D. Marcum, Mary Grace Weiss, Ashleigh E. Stoddart, Miriam Amiram, Arnaz K. Ranji Charna, Jaymin R. Patel, Farren J. Isaacs, Neil L. Kelleher, Seok Hoon Hong, Michael C. Jewett^*

^*Corresponding author for this work

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

165 Scopus citations

Abstract

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation (≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

Original language	English (US)
Article number	1203
Journal	Nature communications
Volume	9
Issue number	1
DOIs	https://doi.org/10.1038/s41467-018-03469-5
State	Published - Dec 1 2018

Funding

This work was supported by the Office of Naval Research (N00014-11-1-0363), the Defense Advanced Research Projects Agency (N66001-12-C-4211), the David and Lucile Packard Foundation, the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the Air Force Research laboratory (FA8650-15-2-5518), the Army Research Office (W911NF-11-1-0445 and W911NF-16-1-0372), the National Science Foundation (MCB-1716766), the National Institutes of Health (NIH-MSTP-TG-T32GM07205), the Dreyfus Teacher-Scholar program, and the Arnold and Mabel Beckman Foundation. 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. R.W.M. is a recipient of the Ford Foundation Fellowship and NSF Graduate Fellowship. B.J.D. is a recipient of the NSF Graduate Fellowship. J. K. is an NDSEG Fellow. We thank Professor James R. Swartz for providing pK7-catGM-CSF plasmid, Professor Bradley Bundy for providing pY71 plasmids, and Professor Peter G. Schultz for providing the pEVOL plasmid. We thank Dr Rui Gan for constructing the pDAK-pAzFRS plasmid. The U.S. 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 Air Force Research Laboratory, the Department of Defense, or the U.S. Government.

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1038/s41467-018-03469-5

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Martin, R. W., Des Soye, B. J., Kwon, Y. C., Kay, J., Davis, R. G., Thomas, P. M., Majewska, N. I., Chen, C. X., Marcum, R. D., Weiss, M. G., Stoddart, A. E., Amiram, M., Ranji Charna, A. K., Patel, J. R., Isaacs, F. J., Kelleher, N. L., Hong, S. H., & Jewett, M. C. (2018). Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Nature communications, 9(1), Article 1203. https://doi.org/10.1038/s41467-018-03469-5

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title = "Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids",

abstract = "Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation (≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.",

author = "Martin, {Rey W.} and {Des Soye}, {Benjamin J.} and Kwon, {Yong Chan} and Jennifer Kay and Davis, {Roderick G.} and Thomas, {Paul M.} and Majewska, {Natalia I.} and Chen, {Cindy X.} and Marcum, {Ryan D.} and Weiss, {Mary Grace} and Stoddart, {Ashleigh E.} and Miriam Amiram and {Ranji Charna}, {Arnaz K.} and Patel, {Jaymin R.} and Isaacs, {Farren J.} and Kelleher, {Neil L.} and Hong, {Seok Hoon} and Jewett, {Michael C.}",

note = "Funding Information: This work was supported by the Office of Naval Research (N00014-11-1-0363), the Defense Advanced Research Projects Agency (N66001-12-C-4211), the David and Lucile Packard Foundation, the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the Air Force Research laboratory (FA8650-15-2-5518), the Army Research Office (W911NF-11-1-0445 and W911NF-16-1-0372), the National Science Foundation (MCB-1716766), the National Institutes of Health (NIH-MSTP-TG-T32GM07205), the Dreyfus Teacher-Scholar program, and the Arnold and Mabel Beckman Foundation. 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. R.W.M. is a recipient of the Ford Foundation Fellowship and NSF Graduate Fellowship. B.J.D. is a recipient of the NSF Graduate Fellowship. J. K. is an NDSEG Fellow. We thank Professor James R. Swartz for providing pK7-catGM-CSF plasmid, Professor Bradley Bundy for providing pY71 plasmids, and Professor Peter G. Schultz for providing the pEVOL plasmid. We thank Dr Rui Gan for constructing the pDAK-pAzFRS plasmid. The U.S. 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 Air Force Research Laboratory, the Department of Defense, or the U.S. Government. Publisher Copyright: {\textcopyright} 2018 The Author(s).",

year = "2018",

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doi = "10.1038/s41467-018-03469-5",

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Martin, RW, Des Soye, BJ, Kwon, YC, Kay, J, Davis, RG, Thomas, PM, Majewska, NI, Chen, CX, Marcum, RD, Weiss, MG, Stoddart, AE, Amiram, M, Ranji Charna, AK, Patel, JR, Isaacs, FJ, Kelleher, NL, Hong, SH & Jewett, MC 2018, 'Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids', Nature communications, vol. 9, no. 1, 1203. https://doi.org/10.1038/s41467-018-03469-5

TY - JOUR

T1 - Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids

AU - Martin, Rey W.

AU - Des Soye, Benjamin J.

AU - Kwon, Yong Chan

AU - Kay, Jennifer

AU - Davis, Roderick G.

AU - Thomas, Paul M.

AU - Majewska, Natalia I.

AU - Chen, Cindy X.

AU - Marcum, Ryan D.

AU - Weiss, Mary Grace

AU - Stoddart, Ashleigh E.

AU - Amiram, Miriam

AU - Ranji Charna, Arnaz K.

AU - Patel, Jaymin R.

AU - Isaacs, Farren J.

AU - Kelleher, Neil L.

AU - Hong, Seok Hoon

AU - Jewett, Michael C.

N1 - Funding Information: This work was supported by the Office of Naval Research (N00014-11-1-0363), the Defense Advanced Research Projects Agency (N66001-12-C-4211), the David and Lucile Packard Foundation, the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust, the Air Force Research laboratory (FA8650-15-2-5518), the Army Research Office (W911NF-11-1-0445 and W911NF-16-1-0372), the National Science Foundation (MCB-1716766), the National Institutes of Health (NIH-MSTP-TG-T32GM07205), the Dreyfus Teacher-Scholar program, and the Arnold and Mabel Beckman Foundation. 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. R.W.M. is a recipient of the Ford Foundation Fellowship and NSF Graduate Fellowship. B.J.D. is a recipient of the NSF Graduate Fellowship. J. K. is an NDSEG Fellow. We thank Professor James R. Swartz for providing pK7-catGM-CSF plasmid, Professor Bradley Bundy for providing pY71 plasmids, and Professor Peter G. Schultz for providing the pEVOL plasmid. We thank Dr Rui Gan for constructing the pDAK-pAzFRS plasmid. The U.S. 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 Air Force Research Laboratory, the Department of Defense, or the U.S. Government. Publisher Copyright: © 2018 The Author(s).

PY - 2018/12/1

Y1 - 2018/12/1

N2 - Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation (≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

AB - Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation (≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

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Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids

Abstract

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