Abstract
The selenium moiety in selenocysteine (Sec) imparts enhanced chemical properties to this amino acid and ultimately the protein in which it is inserted. These characteristics are attractive for designing highly active enzymes or extremely stable proteins and studying protein folding or electron transfer, to name a few. There are also 25 human selenoproteins, of which many are essential for our survival. The ability to create or study these selenoproteins is significantly hindered by the inability to easily produce them. Engineering translation has yielded simpler systems to facilitate site-specific insertion of Sec; however, Ser misincorporation remains problematic. Therefore, we have designed two Sec-specific reporters which promote high-throughput screening of Sec translation systems to overcome this barrier. This protocol outlines the workflow to engineer these Sec-specific reporters, with the application to any gene of interest and the ability to transfer this strategy to any organism.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Meng K, Chung CZ, Söll D, Krahn N (2022) Unconventional genetic code systems in archaea. Front Microbiol 13:1007832. https://doi.org/10.3389/fmicb.2022.1007832
Chung CZ, Krahn N (2022) The selenocysteine toolbox: a guide to studying the 21st amino acid. Arch Biochem Biophys 730:109421. https://doi.org/10.1016/j.abb.2022.109421
Mukai T, Sevostyanova A, Suzuki T, Fu X, Söll D (2018) A facile method for producing selenocysteine-containing proteins. Angew Chem Int Ed Engl 57(24):7215–7219. https://doi.org/10.1002/anie.201713215
Aldag C, Bröcker MJ, Hohn MJ, Prat L, Hammond G, Plummer A, Söll D (2013) Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation. Angew Chem Int Ed Engl 52(5):1441–1445. https://doi.org/10.1002/anie.201207567
Thyer R, Robotham SA, Brodbelt JS, Ellington AD (2015) Evolving tRNASec for efficient canonical incorporation of selenocysteine. J Am Chem Soc 137(1):46–49. https://doi.org/10.1021/ja510695g
Miller C, Bröcker MJ, Prat L, Ip K, Chirathivat N, Feiock A, Veszpremi M, Söll D (2015) A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro. FEBS Lett 589(17):2194–2199. https://doi.org/10.1016/j.febslet.2015.06.039
Majiduddin FK, Palzkill T (2003) Amino acid sequence requirements at residues 69 and 238 for the SME-1 beta-lactamase to confer resistance to beta-lactam antibiotics. Antimicrob Agents Chemother 47(3):1062–1067. https://doi.org/10.1128/AAC.47.3.1062-1067.2003
Thyer R, Shroff R, Klein DR, d’Oelsnitz S, Cotham VC, Byrom M, Brodbelt JS, Ellington AD (2018) Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains. Nat Biotechnol 36(7):624–631. https://doi.org/10.1038/nbt.4154
Chung CZ, Krahn N, Crnković A, Söll D (2022) Intein-based design expands diversity of selenocysteine reporters. J Mol Biol 434(8):167199. https://doi.org/10.1016/j.jmb.2021.167199
Shah NH, Muir TW (2014) Inteins: nature’s gift to protein chemists. Chem Sci 5(1):446–461. https://doi.org/10.1039/C3SC52951G
Mills KV, Johnson MA, Perler FB (2014) Protein splicing: how inteins escape from precursor proteins. J Biol Chem 289(21):14498–14505. https://doi.org/10.1074/jbc.R113.540310
Appleby-Tagoe JH, Thiel IV, Wang Y, Wang Y, Mootz HD, Liu XQ (2011) Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution. J Biol Chem 286(39):34440–34447. https://doi.org/10.1074/jbc.M111.277350
Stevens AJ, Sekar G, Shah NH, Mostafavi AZ, Cowburn D, Muir TW (2017) A promiscuous split intein with expanded protein engineering applications. Proc Natl Acad Sci U S A 114(32):8538–8543. https://doi.org/10.1073/pnas.1701083114
Marshall CJ, Grosskopf VA, Moehling TJ, Tillotson BJ, Wiepz GJ, Abbott NL, Raines RT, Shusta EV (2015) An evolved Mxe GyrA intein for enhanced production of fusion proteins. ACS Chem Biol 10(2):527–538. https://doi.org/10.1021/cb500689g
Phillips GJ, Park SK, Huber D (2000) High copy number plasmids compatible with commonly used cloning vectors. BioTechniques 28(3):400–408. https://doi.org/10.2144/00283bm02
Tharp JM, Ad O, Amikura K, Ward FR, Garcia EM, Cate JHD, Schepartz A, Söll D (2020) Initiation of protein synthesis with non-canonical amino acids in vivo. Angew Chem Int Ed Engl 59(8):3122–3126. https://doi.org/10.1002/anie.201914671
Acknowledgements
We thank Oscar Vargas-Rodriguez and Kexin Meng for experimental advice and helpful discussions.
This work was supported by grants from the National Institute of General Medical Sciences (R35GM122560-05S1 to D.S.) and, for the genetic studies, the Department of Energy Office of Basic Energy Sciences (DE-FG0298ER2031 to D.S.). Christina Z. Chung holds a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada (NSERC).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Chung, C.Z., Söll, D., Krahn, N. (2023). Creating Selenocysteine-Specific Reporters Using Inteins. In: Tsai, YH., Elsässer, S.J. (eds) Genetically Incorporated Non-Canonical Amino Acids. Methods in Molecular Biology, vol 2676. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3251-2_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3251-2_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3250-5
Online ISBN: 978-1-0716-3251-2
eBook Packages: Springer Protocols