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Isolating and Engineering Fluorescence-Activating Proteins Using Yeast Surface Display

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Yeast Surface Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2491))

Abstract

This protocol describes the workflow to isolate and engineer fluorescence-activating proteins by yeast surface display. Fluorescence-activating proteins are an emerging class of fluorescent chemogenetic reporters for monitoring gene expression and protein localization in living cells and organisms. They become fluorescent upon binding exogenously applied fluorogenic organic dyes. Efficient fluorescence-activating proteins can be selected from yeast-displayed libraries by iterative rounds of fluorescence-activated cell sorting. The overall strategy is described, as well as a strategy for characterizing the affinity and spectroscopic properties of the selected clones.

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References

  1. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557

    Article  CAS  Google Scholar 

  2. Boder ET, Wittrup KD (2000) Yeast surface display for directed evolution of protein expression, affinity, and stability. In: Thorner J, Emr SD, Abelson JN (eds) Methods in enzymology. Academic Press, Cambridge, pp 430–444

    Google Scholar 

  3. Chen I, Dorr BM, Liu DR (2011) A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci U S A 108:11399–11404

    Article  CAS  Google Scholar 

  4. Boder ET, Raeeszadeh-Sarmazdeh M, Price JV (2012) Engineering antibodies by yeast display. Arch Biochem Biophys 526:99–106

    Article  CAS  Google Scholar 

  5. Szent-Gyorgyi C, Schmidt BF, Creeger Y, Fisher GW, Zakel KL, Adler S, Fitzpatrick JAJ, Woolford CA, Yan Q, Vasilev KV, Berget PB, Bruchez MP, Jarvik JW, Waggoner A (2008) Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat Biotechnol 26:235–240

    Article  CAS  Google Scholar 

  6. Gautier A, Tebo AG (2020) Sensing cellular biochemistry with fluorescent chemical–genetic hybrids. Curr Opin Chem Biol 57:58–64

    Article  CAS  Google Scholar 

  7. Broch F, Gautier A (2020) Illuminating cellular biochemistry: Fluorogenic Chemogenetic biosensors for biological imaging. ChemPlusChem 85:1487–1497

    Article  CAS  Google Scholar 

  8. Ben Aissa H, Gautier A (2020) Engineering glowing Chemogenetic hybrids for spying on cells. Eur J Org Chem 2020:5637–5646

    Article  Google Scholar 

  9. Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z, Revyakin A, Patel R, Macklin JJ, Normanno D, Singer RH, Lionnet T, Lavis LD (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods 12:244–250

    Article  CAS  Google Scholar 

  10. Grimm JB, Muthusamy AK, Liang Y, Brown TA, Lemon WC, Patel R, Lu R, Macklin JJ, Keller PJ, Ji N, Lavis LD (2017) A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat Methods 14:987–994

    Article  CAS  Google Scholar 

  11. Wang L, Tran M, D’Este E, Roberti J, Koch B, Xue L, Johnsson K (2020) A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy. Nat Chem 12:165–172

    Article  CAS  Google Scholar 

  12. Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333:642–646

    Article  CAS  Google Scholar 

  13. Song W, Filonov GS, Kim H, Hirsch M, Li X, Moon JD, Jaffrey SR (2017) Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex. Nat Chem Biol 13:1187–1194

    Article  CAS  Google Scholar 

  14. Chen X, Zhang D, Su N, Bao B, Xie X, Zuo F, Yang L, Wang H, Jiang L, Lin Q, Fang M, Li N, Hua X, Chen Z, Bao C, Xu J, Du W, Zhang L, Zhao Y, Zhu L, Loscalzo J, Yang Y (2019) Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat Biotechnol 137:1287–1293

    Article  Google Scholar 

  15. Plamont M-A, Billon-Denis E, Maurin S, Gauron C, Pimenta FM, Specht CG, Shi J, Quérard J, Pan B, Rossignol J (2016) Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo. Proc Natl Acad Sci U S A 113:497–502

    Article  CAS  Google Scholar 

  16. Li C, Tebo AG, Thauvin M, Plamont M-A, Volovitch M, Morin X, Vriz S, Gautier A (2020) A far-red emitting fluorescent Chemogenetic reporter for in vivo molecular imaging. Angew Chem Int Ed 59:17917–17923

    Article  CAS  Google Scholar 

  17. Benaissa H, Ounoughi K, Aujard I, Fischer E, Goïame R, Nguyen J, Tebo AG, Li C, Saux TL, Danglot L, Pietrancosta N, Morin X, Jullien L, Gautier A (2021) Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging Nat Commun 12:6989

    Google Scholar 

  18. Tebo AG, Moeyaert B, Thauvin M, Carlon-Andres I, Böken D, Volovitch M, Padilla-Parra S, Dedecker P, Vriz S, Gautier A (2020) Orthogonal fluorescent chemogenetic reporters for multicolor imaging. Nat Chem Biol 17:30–38

    Article  Google Scholar 

  19. Streett HE, Kalis KM, Papoutsakis ET (2019) A strongly fluorescing anaerobic reporter and protein-tagging system for clostridium organisms based on the fluorescence-activating and absorption-shifting tag protein (FAST). Appl Environ Microbiol 85:e00622–e00619

    Article  CAS  Google Scholar 

  20. Charubin K, Modla S, Caplan JL, Papoutsakis ET (2020) Interspecies microbial fusion and large-scale exchange of cytoplasmic proteins and RNA in a syntrophic clostridium coculture. mBio 11:e02030–e02020

    Article  CAS  Google Scholar 

  21. Flaiz M, Ludwig G, Bengelsdorf FR, Dürre P (2021) Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum. Biotechnol Biofuels 14:117

    Article  CAS  Google Scholar 

  22. Peron-Cane C, Fernandez J-C, Leblanc J, Wingertsmann L, Gautier A, Desprat N, Lebreton A (2020) Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of listeria monocytogenes in epithelial cells. PLoS Pathog 16:e1009001

    Article  CAS  Google Scholar 

  23. Chekli Y, Peron-Cane C, Dell’Arciprete D, Allemand J-F, Li C, Ghigo J-M, Gautier A, Lebreton A, Desprat N, Beloin C (2020) Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST. Sci Rep 10:15791

    Article  CAS  Google Scholar 

  24. Tebo AG, Pimenta FM, Zoumpoulaki M, Kikuti C, Sirkia H, Plamont M-A, Houdusse A, Gautier A (2018) Circularly permuted Fluorogenic proteins for the Design of Modular Biosensors. ACS Chem Biol 13:2392–2397

    Article  CAS  Google Scholar 

  25. Tebo AG, Gautier A (2019) A split fluorescent reporter with rapid and reversible complementation. Nat Commun 10:2822

    Article  Google Scholar 

  26. Smith EM, Gautier A, Puchner EM (2019) Single-molecule localization microscopy with the fluorescence-activating and absorption-shifting tag (FAST) system. ACS Chem Biol 14:1115–1120

    Article  CAS  Google Scholar 

  27. Venkatachalapathy M, Belapurkar V, Jose M, Gautier A, Nair D (2019) Live cell super resolution imaging by radial fluctuations using fluorogen binding tags. Nanoscale 11:3626–3632

    Article  CAS  Google Scholar 

  28. Brudler R, Meyer TE, Genick UK, Devanathan S, Woo TT, Millar DP, Gerwert K, Cusanovich MA, Tollin G, Getzoff ED (2000) Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein. Biochemistry (Mosc) 39:13478–13486

    Article  CAS  Google Scholar 

  29. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345

    Article  CAS  Google Scholar 

  30. Gietz RD, Schiestl RH (2007) Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:38–41

    Article  CAS  Google Scholar 

  31. Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346

    Article  CAS  Google Scholar 

  32. Stemmer WP (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370:389–391

    Article  CAS  Google Scholar 

  33. Crameri A, Raillard S-A, Bermudez E, Stemmer WPC (1998) DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391:288–291

    Article  CAS  Google Scholar 

  34. Coco WM, Levinson WE, Crist MJ, Hektor HJ, Darzins A, Pienkos PT, Squires CH, Monticello DJ (2001) DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat Biotechnol 19:354–359

    Article  CAS  Google Scholar 

  35. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1:755–768

    Article  CAS  Google Scholar 

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Acknowledgments

This chapter contains protocols that have been developed and improved over the years and the projects. We warmly and deeply thank Marie-Aude Plamont, Alison G. Tebo, Chenge Li, Emmanuelle Billon-Denis, and Frederico M. Pimenta for their immense contributions to the engineering and characterization of FAST and its variants. This work has been supported by the European Research Council (ERC-2016-CoG-724705 FLUOSWITCH) and a prematuration grant from PSL University and QLife. LEH thanks the Ecole Normale Supérieure for PhD funding.

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Author notes

  1. Lina El Hajji and Hela Benaissa contributed equally to this work.

Authors and Affiliations

  1. Sorbonne Université, École Normale Supérieure, Université PSL, CNRS, Laboratoire des biomolécules, LBM, Paris, France

    Lina El Hajji, Hela Benaissa & Arnaud Gautier

  2. Institut Universitaire de, Paris, France

    Arnaud Gautier

Authors
  1. Lina El Hajji
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  2. Hela Benaissa
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  3. Arnaud Gautier
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Corresponding author

Correspondence to Arnaud Gautier .

Editor information

Editors and Affiliations

  1. Department of Chemistry, Institute of Biochemistry, University of Natural Resources and Life Sciences, Vienna, Austria

    Michael W. Traxlmayr

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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El Hajji, L., Benaissa, H., Gautier, A. (2022). Isolating and Engineering Fluorescence-Activating Proteins Using Yeast Surface Display. In: Traxlmayr, M.W. (eds) Yeast Surface Display. Methods in Molecular Biology, vol 2491. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2285-8_25

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  • DOI: https://doi.org/10.1007/978-1-0716-2285-8_25

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2284-1

  • Online ISBN: 978-1-0716-2285-8

  • eBook Packages: Springer Protocols

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