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Selective and Site-Specific Incorporation of Nonstandard Amino Acids Within Proteins for Therapeutic Applications

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Therapeutic Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2720))

Abstract

The incorporation of nonstandard amino acids (nsAAs) within protein sequences has broadened the chemical functionalities available for use in the study, prevention, or treatment of disease. The ability to genetically encode the introduction of nsAAs at precise sites of target recombinant proteins has enabled numerous applications such as bioorthogonal conjugation, thrombin inhibition, intrinsic biological containment of live organisms, and immunochemical termination of self-tolerance. Genetic systems that perform critical steps in enabling nsAA incorporation are known as orthogonal translation systems or orthogonal aminoacyl-tRNA synthetase/tRNA pairs. In Escherichia coli, several of these have been designed to accept novel nsAAs. Certain endogenous proteins, codon context, and standard amino acid concentrations can affect the yield of recombinant protein, the rate of nsAA incorporation within off-target proteins, and the rate of misincorporation due to near-cognate suppression or misacylation of orthogonal tRNA with standard amino acids. As a result, a significant body of work has been performed in engineering the E. coli genome to alleviate these issues. Here, we describe common methods applicable to nsAA incorporation within proteins in E. coli for sufficient purity and characterization for downstream therapeutic applications.

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Acknowledgments

This work was supported by the National Science Foundation (NSF CBET #2032243).

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA

    Neil D. Butler & Aditya M. Kunjapur

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  1. Neil D. Butler
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  2. Aditya M. Kunjapur
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Corresponding author

Correspondence to Aditya M. Kunjapur .

Editor information

Editors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA

    Millicent O. Sullivan

  2. Department of Molecular Genetics and Microbiology School of Medicine, University of New Mexico, Albuquerque, NM, USA

    Bryce Chackerian

  3. Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA

    Wilfred Chen

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Butler, N.D., Kunjapur, A.M. (2024). Selective and Site-Specific Incorporation of Nonstandard Amino Acids Within Proteins for Therapeutic Applications. In: Sullivan, M.O., Chackerian, B., Chen, W. (eds) Therapeutic Proteins. Methods in Molecular Biology, vol 2720. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3469-1_3

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  • DOI: https://doi.org/10.1007/978-1-0716-3469-1_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3468-4

  • Online ISBN: 978-1-0716-3469-1

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