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 and Michael C. Jewett ()
Additional contact information
Rey W. Martin: Northwestern University
Benjamin J. Des Soye: Northwestern University
Yong-Chan Kwon: Northwestern University
Jennifer Kay: Northwestern University
Roderick G. Davis: Northwestern University
Paul M. Thomas: Northwestern University
Natalia I. Majewska: Northwestern University
Cindy X. Chen: Northwestern University
Ryan D. Marcum: Northwestern University
Mary Grace Weiss: Northwestern University
Ashleigh E. Stoddart: Northwestern University
Miriam Amiram: Yale University
Arnaz K. Ranji Charna: Northwestern University
Jaymin R. Patel: Yale University
Farren J. Isaacs: Yale University
Neil L. Kelleher: Northwestern University
Seok Hoon Hong: Illinois Institute of Technology
Michael C. Jewett: Northwestern University

Nature Communications, 2018, vol. 9, issue 1, 1-9

Abstract: 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.

Date: 2018
References: Add references at CitEc
Citations: View citations in EconPapers (4)

Downloads: (external link)
https://www.nature.com/articles/s41467-018-03469-5 Abstract (text/html)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-03469-5

Ordering information: This journal article can be ordered from
https://www.nature.com/ncomms/

DOI: 10.1038/s41467-018-03469-5

Access Statistics for this article

Nature Communications is currently edited by Nathalie Le Bot, Enda Bergin and Fiona Gillespie

More articles in Nature Communications from Nature
Bibliographic data for series maintained by Sonal Shukla () and Springer Nature Abstracting and Indexing ().