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 journalArticle

59 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 languageEnglish (US)
Article number1203
JournalNature communications
Volume9
Issue number1
DOIs
StatePublished - Dec 1 2018

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)

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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), [1203]. https://doi.org/10.1038/s41467-018-03469-5