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In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins
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    In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins
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    • Yi Liu
      Yi Liu
      Interdisciplinary Biological Sciences Graduate Program,  Department of Chemical and Biological Engineering  and  Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
      More by Yi Liu
    • Roderick G. Davis
      Roderick G. Davis
      Chemistry of Life Processes Institute  and  Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    • Paul M. Thomas
      Paul M. Thomas
      Chemistry of Life Processes Institute  and  Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    • Neil L. Kelleher
      Neil L. Kelleher
      Interdisciplinary Biological Sciences Graduate Program,  Chemistry of Life Processes Institute  and  Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    • Michael C. Jewett*
      Michael C. Jewett
      Interdisciplinary Biological Sciences Graduate Program,  Department of Chemical and Biological Engineering,  Chemistry of Life Processes Institute,  Robert H. Lurie Comprehensive Cancer Center,  Simpson Querrey Institute  and  Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
      *Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208. Phone: 847-467-5007. Email: [email protected]
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    Biochemistry

    Cite this: Biochemistry 2021, 60, 3, 161–169
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    https://doi.org/10.1021/acs.biochem.0c00829
    Published January 11, 2021
    Copyright © 2021 American Chemical Society

    Abstract

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    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.

    Copyright © 2021 American Chemical Society

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    Supporting Information

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    Cited By

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    This article is cited by 12 publications.

    1. Alan Costello, Alexander A. Peterson, Pei-Hsin Chen, Rustam Bagirzadeh, David L. Lanster, Ahmed H. Badran. Genetic Code Expansion History and Modern Innovations. Chemical Reviews 2024, Article ASAP.
    2. Briana R. Lino, Sean J. Williams, Michelle E. Castor, James A. Van Deventer. Reaching New Heights in Genetic Code Manipulation with High Throughput Screening. Chemical Reviews 2024, Article ASAP.
    3. Jessica A. Willi, Ashty S. Karim, Michael C. Jewett. Cell-Free Translation Quantification via a Fluorescent Minihelix. ACS Synthetic Biology 2024, 13 (7) , 2253-2259. https://doi.org/10.1021/acssynbio.4c00266
    4. Amal Seffouh, Rainer Nikolay, Joaquin Ortega. Critical steps in the assembly process of the bacterial 50S ribosomal subunit. Nucleic Acids Research 2024, 52 (8) , 4111-4123. https://doi.org/10.1093/nar/gkae199
    5. Kelly Zhi Qi Zhou, Richard Obexer. Non‐Canonical Amino Acids for Engineering Peptides and Proteins with new Functions. Israel Journal of Chemistry 2024, 296 https://doi.org/10.1002/ijch.202400006
    6. Carlos A. Elena-Real, Annika Urbanek, Xamuel L. Lund, Anna Morató, Amin Sagar, Aurélie Fournet, Alejandro Estaña, Tracy Bellande, Frédéric Allemand, Juan Cortés, Nathalie Sibille, Ronald Melki, Pau Bernadó. Multi-site-specific isotopic labeling accelerates high-resolution structural investigations of pathogenic huntingtin exon-1. Structure 2023, 31 (6) , 644-650.e5. https://doi.org/10.1016/j.str.2023.04.003
    7. Kavan Gor, Olivier Duss. Emerging Quantitative Biochemical, Structural, and Biophysical Methods for Studying Ribosome and Protein–RNA Complex Assembly. Biomolecules 2023, 13 (5) , 866. https://doi.org/10.3390/biom13050866
    8. Xiyu Dong, Lili K Doerfel, Kai Sheng, Jessica N Rabuck-Gibbons, Anna M Popova, Dmitry Lyumkis, James R Williamson. Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways. Nucleic Acids Research 2023, 51 (6) , 2862-2876. https://doi.org/10.1093/nar/gkad082
    9. Kanghun Lee, Jessica A. Willi, Namjin Cho, Inseon Kim, Michael C. Jewett, Joongoo Lee. Cell-free Biosynthesis of Peptidomimetics. Biotechnology and Bioprocess Engineering 2023, 494 https://doi.org/10.1007/s12257-022-0268-5
    10. Yuhui Du, Li Li, Yue Zheng, Jiaheng Liu, Julia Gong, Zekai Qiu, Yanni Li, Jianjun Qiao, Yi-Xin Huo, . Incorporation of Non-Canonical Amino Acids into Antimicrobial Peptides: Advances, Challenges, and Perspectives. Applied and Environmental Microbiology 2022, 88 (23) https://doi.org/10.1128/aem.01617-22
    11. Ilamaran Meganathan, Mohandass Pachaiyappan, Mayilvahanan Aarthy, Janani Radhakrishnan, Smriti Mukherjee, Ganesh Shanmugam, Jingjing You, Niraikulam Ayyadurai. Recombinant and genetic code expanded collagen-like protein as a tailorable biomaterial. Materials Horizons 2022, 9 (11) , 2698-2721. https://doi.org/10.1039/D2MH00652A
    12. Camila Kofman, Joongoo Lee, Michael C. Jewett. Engineering molecular translation systems. Cell Systems 2021, 12 (6) , 593-607. https://doi.org/10.1016/j.cels.2021.04.001

    Biochemistry

    Cite this: Biochemistry 2021, 60, 3, 161–169
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.biochem.0c00829
    Published January 11, 2021
    Copyright © 2021 American Chemical Society

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