Skip to main content
SpringerLink
Log in

High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins

  • Protocol
  • First Online:
Targeting Enzymes for Pharmaceutical Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2089))

Abstract

Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure–activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E, Carver T, Asel E, Springer BA, Lane P, Salemme FR (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6(6):429–440. https://doi.org/10.1177/108705710100600609

    Article  CAS  PubMed  Google Scholar 

  2. Moreau MJ, Morin I, Askin S, Cooper A, Moreland NJ, Vasudevan SG, Schaeffer PM (2012) Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins. RSC Adv 2:11892–11900

    Article  CAS  Google Scholar 

  3. Moreau MJ, Schaeffer PM (2013) Dissecting the salt dependence of the Tus-Ter protein-DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus. Mol BioSyst 9(12):3146–3154. https://doi.org/10.1039/c3mb70426b

    Article  CAS  PubMed  Google Scholar 

  4. Askin SP, Bond TEH, Schaeffer PM (2016) Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase. Anal Methods UK 8(2):418–424

    Article  CAS  Google Scholar 

  5. Bond TEH, Sorenson AE, Schaeffer PM (2017) Functional characterisation of Burkholderia pseudomallei biotin protein ligase: a toolkit for anti-melioidosis drug development. Microbiol Res 199:40–48. https://doi.org/10.1016/j.micres.2017.03.007

    Article  CAS  PubMed  Google Scholar 

  6. Bond TEH, Sorenson AE, Schaeffer PM (2017) A green fluorescent protein-based assay for high-throughput ligand-binding studies of a mycobacterial biotin protein ligase. Microbiol Res 205:35–39. https://doi.org/10.1016/j.micres.2017.08.014

    Article  CAS  PubMed  Google Scholar 

  7. Askin S, Bond TEH, Sorenson AE, Moreau MJJ, Antony H, Davis RA, Schaeffer PM (2018) Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors. Chem Commun (Camb) 54(14):1738–1741. https://doi.org/10.1039/c8cc00090e

    Article  CAS  Google Scholar 

  8. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, vol 3, 4th edn. Cold Springs Hoarbour Laboratory Press, New York

    Google Scholar 

  9. Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A 82(4):1074–1078

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dubendorff JW, Studier FW (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219(1):45–59

    Article  CAS  PubMed  Google Scholar 

  11. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1):207–234

    Article  CAS  PubMed  Google Scholar 

  12. Moreau MJ, Morin I, Schaeffer PM (2010) Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system. Mol BioSyst 6(7):1285–1292. https://doi.org/10.1039/c002001j

    Article  CAS  PubMed  Google Scholar 

  13. Morin I, Schaeffer PM (2012) Combining RNA-DNA swapping and quantitative polymerase chain reaction for the detection of influenza A nucleoprotein. Anal Biochem 420(2):121–126. https://doi.org/10.1016/j.ab.2011.09.009

    Article  CAS  PubMed  Google Scholar 

  14. Ferreira CMH, Pinto ISS, Soares EV, Soares HMVM (2015) (Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions—a review. RSC Adv 5:30989–31003

    Article  CAS  Google Scholar 

  15. Fukuda H, Arai M, Kuwajima K (2000) Folding of green fluorescent protein and the cycle3 mutant. Biochemistry 39(39):12025–12032

    Article  CAS  PubMed  Google Scholar 

  16. Hartnett J, Gracyalny J, Slater MR (2006) The single step (KRX) competent cells: efficient cloning and high protein yields. Promega Notes 94:27–30

    Google Scholar 

  17. Schagat T, Friedman R, Ohana OP, Hartnett J, Slater MR (2008) KRX autoinduction protocol: a convenient method for protein expression. Promega Notes 98:16–18

    CAS  Google Scholar 

  18. Waldo GS, Standish BM, Berendzen J, Terwilliger TC (1999) Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol 17(7):691–695. https://doi.org/10.1038/10904

    Article  CAS  PubMed  Google Scholar 

  19. Hurst MO, Fortenberry RC (2015) Factors affecting the solubility of ionic compounds. Computat Theoret Chem 1069:132–137

    Article  CAS  Google Scholar 

  20. Vagenende V, Yap MG, Trout BL (2009) Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry 48(46):11084–11096. https://doi.org/10.1021/bi900649t

    Article  CAS  PubMed  Google Scholar 

  21. Antony H, Schaeffer PM (2013) A GFP-tagged nucleoprotein-based aggregation assay for anti-influenza drug discovery and antibody development. Analyst 138(20):6073–6080. https://doi.org/10.1039/c3an01041d

    Article  CAS  PubMed  Google Scholar 

  22. Dawson RMC, Elliot DC, Elliot WH, Jones KM (1989) Data for biochemical research, 3rd edn. Oxford University Press, Oxford

    Google Scholar 

  23. Matulis D, Kranz JK, Salemme FR, Todd MJ (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44(13):5258–5266. https://doi.org/10.1021/bi048135v

    Article  CAS  PubMed  Google Scholar 

  24. Cimmperman P, Baranauskiene L, Jachimoviciute S, Jachno J, Torresan J, Michailoviene V, Matuliene J, Sereikaite J, Bumelis V, Matulis D (2008) A quantitative model of thermal stabilization and destabilization of proteins by ligands. Biophys J 95(7):3222–3231. https://doi.org/10.1529/biophysj.108.134973

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Molecular and Cell Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Douglas, QLD, Australia

    Alanna E. Sorenson & Patrick M. Schaeffer

Authors
  1. Alanna E. Sorenson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Patrick M. Schaeffer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Patrick M. Schaeffer .

Editor information

Editors and Affiliations

  1. Department of Biotechnology, Agricultural University of Athens, Athens, Greece

    Nikolaos E. Labrou

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sorenson, A.E., Schaeffer, P.M. (2020). High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins. In: Labrou, N. (eds) Targeting Enzymes for Pharmaceutical Development. Methods in Molecular Biology, vol 2089. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0163-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0163-1_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0162-4

  • Online ISBN: 978-1-0716-0163-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation