Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system
Moreau, Morgane J.J., Morin, Isabelle, and Schaeffer, Patrick M. (2010) Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system. Molecular BioSystems, 6 (7). pp. 1285-1292.
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View at Publisher Website: http://dx.doi.org/10.1039/C002001J
Information about the stability of proteins is paramount to determine their optimal storage or reaction conditions. It is also essential to determine protein stability in high-throughput when screening for new or improved functions of proteins obtained from large mutant libraries. In drug discovery programs, monitoring of ligand-induced stabilization effects can be used to identify lead compounds in high-throughput. These studies require expensive biophysical instrumentation and large quantities of purified proteins. To address these issues, we developed a new method, using GFP as a reporter system to quantify the stability of a protein and its ligand-associated stabilization effects that requires neither special equipment nor extensive purification steps. Here, GFP is fused to a protein of interest (POI) through a linker and is used as a reporter system for protein unfolding and aggregation. The three POIs used in this study include the Ter-binding protein Tus, glycerol kinase and chloramphenicol acetyl transferase. The fluorescent fusion protein is subjected to irreversible thermal denaturation leading to formation of aggregates, which are eliminated by a centrifugation step. The residual fluorescence of the soluble fraction can be directly related to the stability of the POI and can be quantitatively monitored using a fluorescence plate reader. The GFP-based stability assay (GFP-Basta) was able to identify stabilizing compounds and afforded a new quantitative method for the screening and ranking of ligands for three different proteins. These applications are particularly useful for drug discovery, directed evolution, structural and functional genomics.
|Item Type:||Article (Refereed Research - C1)|
|FoR Codes:||06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060101 Analytical Biochemistry @ 100%|
|SEO Codes:||97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%|
|Deposited On:||05 Apr 2011 14:11|
|Last Modified:||18 Oct 2013 01:12|
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