In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins

Yi Liu, Roderick G. Davis, Paul M. Thomas, Neil L. Kelleher, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

Efforts to expand the scope of ribosome-mediated polymerization to incorporate noncanonical amino acids (ncAAs) into peptides and proteins hold promise for creating new classes of enzymes, therapeutics, and materials. Recently, the integrated synthesis, assembly, and translation (iSAT) system was established to construct functional ribosomes in cell-free systems. However, the iSAT system has not been shown to be compatible with genetic code expansion. Here, to address this gap, we develop an iSAT platform capable of manufacturing pure proteins with site-specifically incorporated ncAAs. We first establish an iSAT platform based on extracts from genomically recoded Escherichia coli lacking release factor 1 (RF-1). This permits complete reassignment of the amber codon translation function. Next, we optimize orthogonal translation system components to demonstrate the benefits of genomic RF-1 deletion on incorporation of ncAAs into proteins. Using our optimized platform, we demonstrate high-level, multi-site incorporation of p-acetyl-phenylalanine (pAcF) and p-azido-phenylalanine into superfolder green fluorescent protein (sfGFP). Mass spectrometry analysis confirms the high accuracy of incorporation for pAcF at one, two, and five amber sites in sfGFP. The iSAT system updated for ncAA incorporation sets the stage for investigating ribosomal mutations to better understand the fundamental basis of protein synthesis, manufacturing proteins with new properties, and engineering ribosomes for novel polymerization chemistries.

Original languageEnglish (US)
Pages (from-to)161-169
Number of pages9
JournalBiochemistry
Volume60
Issue number3
DOIs
StatePublished - Jan 26 2021

Funding

This work was supported by the Army Research Office (W911NF-11-1-044, W911NF-18-1-0181, and W911NF-16-1-0372) and the National Science Foundation (MCB-1716766). M.C.J. is a David and Lucile Packard Foundation Fellow. Y.L. is a Medical Scientist Training Program scholar under MSTP NIH T32 Training Grant T32GM008152. This research was also 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, and supported by the Sherman Fairchild Foundation.

ASJC Scopus subject areas

  • Biochemistry

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