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Protein Scaffold-Activated Protein Trans-Splicing in Mammalian Cells

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† Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States
‡ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States
Cite this: J. Am. Chem. Soc. 2013, 135, 20, 7713-7719
Publication Date (Web):April 26, 2013
https://doi.org/10.1021/ja401689b
Copyright © 2013 American Chemical Society
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Abstract

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Conditional protein splicing is a powerful biotechnological tool that can be used to rapidly and post-translationally control the activity of a given protein. Here we demonstrate a novel conditional splicing system in which a genetically encoded protein scaffold induces the splicing and activation of an enzyme in mammalian cells. In this system the protein scaffold binds to two inactive split intein/enzyme extein protein fragments leading to intein fragment complementation, splicing, and activation of the firefly luciferase enzyme. We first demonstrate the ability of antiparallel coiled-coils (CCs) to mediate splicing between two intein fragments, effectively creating two new split inteins. We then generate and test two versions of the scaffold-induced splicing system using two pairs of CCs. Finally, we optimize the linker lengths of the proteins in the system and demonstrate 13-fold activation of luciferase by the scaffold compared to the activity of negative controls. Our protein scaffold-triggered conditional splicing system is an effective strategy to control enzyme activity using a protein input, enabling enhanced genetic control over protein splicing and the potential creation of splicing-based protein sensors and autoregulatory systems.

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DNA titration of LZA-EE scaffold system, DNA constructs and subparts, DNA transfection amounts, DNA subpart sequences, amino acid sequences of the coiled-coil domains. This material is available free of charge via the Internet at http://pubs.acs.org.

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Cited By

This article is cited by 20 publications.

  1. Robert Chen, Harneet S. Rishi, Vladimir Potapov, Masaki R. Yamada, Vincent J. Yeh, Thomas Chow, Celia L. Cheung, Austin T. Jones, Terry D. Johnson, Amy E. Keating, William C. DeLoache, and John E. Dueber . A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy. ACS Synthetic Biology 2015, 4 (11) , 1205-1216. DOI: 10.1021/acssynbio.5b00060.
  2. Nichole M. Daringer, Rachel M. Dudek, Kelly A. Schwarz, and Joshua N. Leonard . Modular Extracellular Sensor Architecture for Engineering Mammalian Cell-based Devices. ACS Synthetic Biology 2014, 3 (12) , 892-902. DOI: 10.1021/sb400128g.
  3. Tina Lebar, Duško Lainšček, Estera Merljak, Jana Aupič, Roman Jerala. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nature Chemical Biology 2020, 26 DOI: 10.1038/s41589-019-0443-y.
  4. Brendan J Hussey, David R McMillen. Programmable T7-based synthetic transcription factors. Nucleic Acids Research 2018, 46 (18) , 9842-9854. DOI: 10.1093/nar/gky785.
  5. Ashwin Lahiry, Yamin Fan, Samuel D Stimple, Mitch Raith, David W Wood. Inteins as tools for tagless and traceless protein purification. Journal of Chemical Technology & Biotechnology 2018, 93 (7) , 1827-1835. DOI: 10.1002/jctb.5415.
  6. Tim Sonntag. A Cassette Approach for the Identification of Intein Insertion Sites. 2017,,, 239-258. DOI: 10.1007/978-1-4939-6451-2_16.
  7. Katarzyna P. Adamala, Daniel A. Martin-Alarcon, Edward S. Boyden. Programmable RNA-binding protein composed of repeats of a single modular unit. Proceedings of the National Academy of Sciences 2016, 113 (19) , E2579-E2588. DOI: 10.1073/pnas.1519368113.
  8. Anton Dobrin, Pratik Saxena, Martin Fussenegger. Synthetic biology: applying biological circuits beyond novel therapies. Integrative Biology 2016, 8 (4) , 409-430. DOI: 10.1039/c5ib00263j.
  9. Jan Lonzarić, Tina Lebar, Andreja Majerle, Mateja Manček-Keber, Roman Jerala. Locked and proteolysis-based transcription activator-like effector (TALE) regulation. Nucleic Acids Research 2016, 44 (3) , 1471-1481. DOI: 10.1093/nar/gkv1541.
  10. Aina Ollé-Vila, Salva Duran-Nebreda, Núria Conde-Pueyo, Raúl Montañez, Ricard Solé. A morphospace for synthetic organs and organoids: the possible and the actual. Integrative Biology 2016, 8 (4) , 485-503. DOI: 10.1039/C5IB00324E.
  11. Robert W. Bradley, Baojun Wang. Designer cell signal processing circuits for biotechnology. New Biotechnology 2015, 32 (6) , 635-643. DOI: 10.1016/j.nbt.2014.12.009.
  12. Yifeng Li. Split-inteins and their bioapplications. Biotechnology Letters 2015, 37 (11) , 2121-2137. DOI: 10.1007/s10529-015-1905-2.
  13. Daniel Demonte, Naiyi Li, Sheldon Park. Postsynthetic Domain Assembly with NpuDnaE and SspDnaB Split Inteins. Applied Biochemistry and Biotechnology 2015, 177 (5) , 1137-1151. DOI: 10.1007/s12010-015-1802-0.
  14. Shimyn Slomovic, James J Collins. DNA sense-and-respond protein modules for mammalian cells. Nature Methods 2015, 12 (11) , 1085-1090. DOI: 10.1038/nmeth.3585.
  15. Heejae Kim, Ka-Hei Siu, Maryam Raeeszadeh-Sarmazdeh, Qing Sun, Qi Chen, Wilfred Chen. Bioengineering strategies to generate artificial protein complexes. Biotechnology and Bioengineering 2015, 112 (8) , 1495-1505. DOI: 10.1002/bit.25637.
  16. Zoltán Kis, Hugo Sant'Ana Pereira, Takayuki Homma, Ryan M. Pedrigi, Rob Krams. Mammalian synthetic biology: emerging medical applications. Journal of The Royal Society Interface 2015, 12 (106) , 20141000. DOI: 10.1098/rsif.2014.1000.
  17. Melissa Cronin, Michael J. Coolbaugh, David Nellis, Jianwei Zhu, David W. Wood, Ruth Nussinov, Buyong Ma. Dynamics differentiate between active and inactive inteins. European Journal of Medicinal Chemistry 2015, 91, 51-62. DOI: 10.1016/j.ejmech.2014.07.094.
  18. Rishi Rakhit, Raul Navarro, Thomas J. Wandless. Chemical Biology Strategies for Posttranslational Control of Protein Function. Chemistry & Biology 2014, 21 (9) , 1238-1252. DOI: 10.1016/j.chembiol.2014.08.011.
  19. Florian Lienert, Jason J. Lohmueller, Abhishek Garg, Pamela A. Silver. Synthetic biology in mammalian cells: next generation research tools and therapeutics. Nature Reviews Molecular Cell Biology 2014, 15 (2) , 95-107. DOI: 10.1038/nrm3738.
  20. Natalya I Topilina, Kenneth V Mills. Recent advances in in vivo applications of intein-mediated protein splicing. Mobile DNA 2014, 5 (1) , 5. DOI: 10.1186/1759-8753-5-5.

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