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Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast

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Biomedical Engineering Technologies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2394))

Abstract

Genetic code expansion has allowed for extraordinary advances in enhancing protein chemical diversity and functionality, but there remains a critical need for understanding and engineering genetic code expansion systems for improved efficiency. Incorporation of noncanonical amino acids (ncAAs) at stop codons provides a site-specific method for introducing unique chemistry into proteins, though often at reduced yields compared to wild-type proteins. A powerful platform for ncAA incorporation supports both the expression and evaluation of chemically diverse proteins for a broad range of applications. In yeast, ncAAs have been used to study dynamic cellular processes such as protein–protein interactions and also allow for exploration of eukaryotic-specific biology such as epigenetics. Furthermore, yeast display is an advantageous technology for engineering and screening the properties of proteins in high throughput. The protocols presented in this chapter describe detailed methods for the yeast-based genetic encoding of ncAAs in proteins intracellularly or on the yeast surface. In addition, methods are presented for modifying proteins on the yeast surface using bioorthogonal chemical reactions and evaluating reaction efficiency. Finally, protocols are included for the preparation of libraries that involve genetic code expansion. Libraries of proteins that contain ncAAs or libraries of the cellular machinery required to encode ncAAs can be constructed and screened in high throughput for many biological and chemical applications. Efficient incorporation of ncAAs facilitates elucidation of fundamental eukaryotic biology and advances tools for enzyme and genome engineering to evolve host cells that are better able to accommodate alternative genetic codes.

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Acknowledgments

We thank our colleagues in the Van Deventer Laboratory for their feedback and insight on these protocols, in particular Rebecca Hershman and Arlinda Rezhdo for their extensive comments. Research that led to the establishment of these protocols was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133471, the National Cancer Institute of the National Institute of Health under Award R21CA214239, the National Science Foundation under award NSF1815022, the Army Research Office under Award Number W911NF-16-1-0175, a Tufts Collaborates research award, and Tufts startup funds (to J.A.V.). J.T.S. was supported in part by an NSF Graduate Research Fellowship (ID: 2016231237). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the National Science Foundation, the Army Research Office, or Tufts University.

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Authors and Affiliations

  1. Chemical and Biological Engineering Department, Tufts University, Medford, MA, USA

    Jessica T. Stieglitz & James A. Van Deventer

  2. Biomedical Engineering Department, Tufts University, Medford, MA, USA

    James A. Van Deventer

Authors
  1. Jessica T. Stieglitz
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  2. James A. Van Deventer
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Corresponding author

Correspondence to James A. Van Deventer .

Editor information

Editors and Affiliations

  1. Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD, USA

    Avraham Rasooly

  2. Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD, USA

    Houston Baker

  3. Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Bethesda, MD, USA

    Miguel R. Ossandon

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Stieglitz, J.T., Van Deventer, J.A. (2022). Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast. In: Rasooly, A., Baker, H., Ossandon, M.R. (eds) Biomedical Engineering Technologies. Methods in Molecular Biology, vol 2394. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1811-0_21

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  • DOI: https://doi.org/10.1007/978-1-0716-1811-0_21

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1810-3

  • Online ISBN: 978-1-0716-1811-0

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