Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system

Abstract

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.

Non‐allosteric enzyme switches possess larger effector‐induced changes in thermodynamic stability than their non‐switch analogs

“…This phenomena arose by an increase in cellular accumulation of the fusion proteins in the presence of maltose–an accumulation arising from a specific interaction between the switch and maltose 6. We termed these switches “phenotypic switches.” The similar ligand‐dependent behavior of engineered fusion proteins have been previously reported in different systems, and these unique characteristics of fusion proteins have been used to develop screening methods for ligand binding and protein stability 7–9. Ligand‐induced upregulation of constitutively active mutant form of β 2 ‐adrenoceptor tagged with the luciferase at the C‐terminal, resulted in elevated levels of luciferase activity, and this system was used to develop high‐throughput assay to monitor ligand binding to G‐protein‐coupled receptor 7.…”

“…Ligand‐induced upregulation of constitutively active mutant form of β 2 ‐adrenoceptor tagged with the luciferase at the C‐terminal, resulted in elevated levels of luciferase activity, and this system was used to develop high‐throughput assay to monitor ligand binding to G‐protein‐coupled receptor 7. In other systems, the fluorescent sensitivity of green fluorescent protein (GFP) to the ligand‐induced folding of proteins fused to its N‐terminus was used to develop high‐throughput method to monitor ligand binding and thermal stability of proteins of interest such as steroid receptor and glycerol kinase 8, 9. The prevalence of the gene fusions that confer switching behavior in the library of nonhomologously recombined genes has important implications for construction and application of protein switching.…”

Stable production of cyanophycinase in Nicotiana benthamiana and its functionality to hydrolyse cyanophycin in the murine intestine

“…The reduced stability of the complete protein was also indicated by the additional bands observed for CPHB‐S‐C, possibly representing degradation products. The N ‐terminal modification may result in a decreased sensitivity to proteases, as suggested for GFP (Moreau et al ., ; Piron et al ., ), ubiquitin (Hondred et al ., ; Jang et al ., ) and elastin‐like proteins (Floss et al ., ; Patel et al ., ). Nevertheless, we cannot exclude the possibility that the increased levels of GFP::CPHB‐S and S‐CPHB‐S are due to altered translational efficiency.…”

Seamless GFP and GFP-Amylase Cloning in Gateway Shuttle Vector, Expression of the Recombinant Proteins inE. ColiandBacillus Megateriumand Assessment of the GFP-Amylase Thermostability

“…Several earlier studies show that both BlA and GFP possess high thermal stability (18,19,25), which makes possible the application of thermolysis extraction for these two proteins. the XylR/pXylA' driven gene expression in B. megaterium cells requires xylose induction, which results in a high background in the subsequent DnS assay of α-amylase activity.…”

“…The fusion of studied proteins with fluorescent proteins is increasingly applied for direct assessment of their heterologous expression and conformation stability under various conditions, including thermal stability (18,29,30). this approach offers an attractive option for comparative characterization of groups of newly cloned genes at a larger scale, including comparison of the expression levels of the corresponding recombinant proteins expressed in common microbial hosts.…”