Abstract
Membrane protein purification often yields rather unstable proteins impeding functional and structural protein characterization. Low protein stability also leads to low purification yields as a result of protein degradation, aggregation, precipitation, and folding instability. It is often required to optimize buffer conditions through numerous iterations of trial and error to improve the homogeneity, stability, and solubility of the protein sample demanding high amounts of purified protein. Therefore we have set up a fast, simple, and high-throughput time-dependent thermostability-based assay at low protein cost to identify protein stabilizing factors to facilitate the handling and characterization of membrane proteins by subsequent structural and functional studies.
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
Olesen C, Picard M, Winther AM, Gyrup C, Morth JP, Oxvig C, Moller JV, Nissen P (2007) The structural basis of calcium transport by the calcium pump. Nature 450:1036–1042
Yao Q, Chen LT, Bigelow DJ (1998) Affinity purification of the Ca-ATPase from cardiac sarcoplasmic reticulum membranes. Protein Expr Purif 13:191–197
Morth JP, Pedersen BP, Toustrup-Jensen MS, Sorensen TL, Petersen J, Andersen JP, Vilsen B, Nissen P (2007) Crystal structure of the sodium-potassium pump. Nature 450:1043–1049
Boivin S, Kozak S, Meijers R (2013) Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif 91:192–206
Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P (2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem 357:289–298
Alexandrov AI, Mileni M, Chien EY, Hanson MA, Stevens RC (2008) Microscale fluorescent thermal stability assay for membrane proteins. Structure 16:351–359
Abts A, Schwarz CKW, Tschapek B, Smits SHJ, Schmitt L (2011) Rational and irrational approaches to convince a protein to crystallize. In: Kolesnikov N, Borisenko E (eds) Modern aspects of bulk crystal and thin film preparation. ISBN p. Chapter 22 497–528
Sonoda Y, Newstead S, Hu NJ, Alguel Y, Nji E, Beis K, Yashiro S, Lee C, Leung J, Cameron AD, Byrne B, Iwata S, Drew D (2011) Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19:17–25
Forneris F, Orru R, Bonivento D, Chiarelli LR, Mattevi A (2009) ThermoFAD, a Thermofluor-adapted flavin ad hoc detection system for protein folding and ligand binding. FEBS J 276:2833–2840
Fan J, Heng J, Dai S, Shaw N, Zhou B, Huang B, He Z, Wang Y, Jiang T, Li X, Liu Z, Wang X, Zhang XC (2011) An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability. Protein Expr Purif 78:6–13
Ayers FC, Warner GL, Smith KL, Lawrence DA (1986) Fluorometric quantitation of cellular and nonprotein thiols. Anal Biochem 154:186–193
Hattori M, Hibbs RE, Gouaux E (2012) A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20:1293–1299
Acknowledgements
This work was supported by the Interuniversity Poles of Attraction of the Belgian Science Policy Office (P7/13) and the Flanders Research Foundation (FWO G044212N, G0B1115N and 1514514N) and the KU Leuven (OT/13/091).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Vandecaetsbeek, I., Vangheluwe, P. (2016). Time-Dependent Protein Thermostability Assay. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3179-8_9
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
eBook Packages: Springer Protocols