The buffer panel consisted of four buffers with different ph. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. Protein stability data was generated for twelve gfptagged proteins including monomeric and multimeric, dnabinding, rnabinding, proteolytic, heatshock and metabolic proteins of escherichia coli. Application of the thermofluor pastry technique for.
Thermal denaturation assays in chemical biology assay. A highthroughput screen for protein stability gareth morgan. Protein stability data was generated for twelve gfptagged proteins including monomeric and multimeric, dnabinding, rnabinding, proteolytic, heatshock and metabolic proteins of escherichia coli, burkholderia pseudomallei, staphylococcus aureus, dengue and influenza h5n1 viruses. Fmdv exists as seven serotypes, each made up by a number of constantly evolving subtypes. How to determine protein stability enzo life sciences. Here, the protein is significantly more stable in 50 mm sodium phosphate buffer, ph 7.
Thermal shift assays or thermofluor provide a highthroughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Thermofad, a thermofluoradapted flavin ad hoc detection. Pipette 2 l of 1 mgml protein per well 2 g of protein per well. Optimization of protein samples for nmr using thermal. A quick and easy approach to identify a buffer composition which increases the stability and possibly the conformational homogeneity of a protein sample is the fluorescence. The thermofluor method can be performed on nearly all qpcr machines, meaning no special instrumentation is necessary, and can be used to validate the quality of protein preparations. Mar 09, 2005 thermofluor a miniaturized highthroughput protein stability assay was used to analyze the linkage between protein thermal stability and ligand binding. Jbscreen thermofluor fundament readytouse tsa for protein stability jena bioscience gmbh lobstedter str. The most common method for measuring protein thermal shifts is differential scanning fluorimetry dsf or thermofluor, which utilizes. Glomelt dye undergoes fluorescence enhancement upon binding to hydrophobic regions of denatured proteins, and therefore can be used to detect protein unfolding or measure thermal stability by performing a thermal shift assay, also called protein thermal shift, differential scanning fluorimetry, or thermofluor assay. Pdf optimization of protein buffer cocktails using thermofluor. Application of the thermofluor pastry technique for improving. Use a multichannel pipette to transfer 40 l of solution into each well of a 96well assay plate. Thermal melting can confirm the protein is correctly folded and provide a measure of how stable it is under a given condition.
The stability and homogeneity of a protein sample is strongly influenced by the composition of the buffer that the protein is in. Isothermal analysis of thermofluor data can readily. Applied biosystems protein thermal shift kits allow users to employ a dye that binds to exposed hydrophobic residues to monitor the thermal stability of proteins using a realtime pcr instrument in order to identify buffer conditions that stabilize the protein of interest or screen libraries of liga. The program offers a number of ways to further analyse the data using simple menudriven choices, as exemplified in fig. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Cs333 jbscreen thermofluor specific hts jena bioscience. Rapid determination of protein stability and ligand. Results the thermofad technique a thermofad analysis requires only 20. With the pcr plate on ice, add the following to each well. Thermofluor is a fluorescencebased thermal stability assay monitoring ligand effects on. Production of proteins well suited for structural studies is inherently difficult and timeconsuming. Thermofluor a miniaturized highthroughput protein stability assay was used to analyze the linkage between protein thermal stability and ligand binding. A thermal shift assay quantifies the change in thermal denaturation temperature of a protein under varying conditions.
One method to perform this analysis is the thermofluor approach. As a consequence, the method can be applied to samples for which aggregation or low stability hinders purification efforts. Highthroughput thermal scanning for protein stability. Undesired overlay of screening of interdependent variables is prevented by strictly categorizing stability screening of proteins into 1. Optimization of protein samples for nmr using thermal shift. Request pdf thermofluorbased highthroughput stability optimization of proteins for structural studies production of proteins well suited for structural studies. The screen encompasses inorganic salts of different nature that were selected taking into consideration the number of successful determined structures on the. Protein stability is a key factor in the crystallization process. Current fmd virus fmdv vaccines are made from chemically inactivated virus and need to contain intact viral capsids to maximize efficacy. Thermofluor is a cheap and fast technique that constitutes an efficient and generic highthroughput method for identification of protein properties being often used to optimize buffer and. Protein thermal shift dye kit thermo fisher scientific. Stabilization of proteins for crystallization how thermofluor can help. Thermodynamic stability of a protein is related to its gibbs free energy of unfolding.
Highthroughput analysis of protein thermal stability. May 28, 20 we present a highthroughput approach to help define experimental formulations that enhance protein stability, which is based on differential scanning fluorimetry dsf. Introduction rubic buffer screen is a set of 96 chemical reagents formulated in ultrapure water at room temperature. Isothermal analysis of thermofluor data can readily provide. The binding of low molecular weight ligands can increase the thermal stability of. The high stability of the renatured protein may be a reminiscent property of sbp from its evolutionary relatedness to the seed storage proteins. Thermofluor stability assay protein stability peak proteins. Accordingly, we write the protein foldingunfolding reaction as a. The crystallization of macromolecules is a complex procedure which is strongly influenced by. Rapid determination of protein stability and ligand binding.
Thermofluor provides a fluorescence readout measurement of thermallyinduced protein melting. A collection and summary on thermofluor assay a corner of. On the contrary, there are factors that are likely to affect mainly solubility, such. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. A thermal shift assay tsa measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer ph or ionic strength, redox potential, or sequence mutation. In this work we examined thermal stability differences between human igg1 and igg4 fab domains containing the same variable regions using the thermofluor assay. Oppf1446 is a dimer which is known to be inactive when dissociated with reducing agents. The protein thermal shift assay was used to screen buffers to identify the ph where the protein reca was most stable figure 2. The use of differential scanning fluorimetry to detect.
Oct 20, 2018 in particular, the biochemical characterization of diseaserelated proteins and their genetic variants that result in misfolding requires systematic determination of protein stability, screening of optimal buffer conditions for biophysical and structural studies, and in some cases, the identification of small molecule chaperones with the. Protein stability is a major concern for biotechnology, food industries, pharmaceuticals, and academic scientists. For a protein that unfolds and refolds rapidly, reversibly, cooperatively, and with a simple, twostate mechanism. Following the thermofluor analysis, the protein was dialyzed into the. The thermal shift assay is a rapid and inexpensive technique that quantifies change in protein. Differential scanning fluorimetry dsf, also known as thermofluor or. Protein stability normally refers to the physical thermodynamic stability, not the chemical stability. Thus, new, more sensitive approaches to quantify capsid stability would be of great value for the production of more stable vaccines and to assess the effect of production conditions on vaccine preparations. Pdf rapid determination of protein stability and ligand. Enhancing recombinant protein quality and yield by protein. Test of the stability of different functional variants carver et al.
Rising temperature causes protein unfolding and the fluorophore sypro orange partitions itself into the melted protein and hence the overall effect is an increase in fluorescence with increasing temperature if a drugligand is included which binds to the protein, the midpoint of the melt curve can shift, indicating stabilising. C an analysis of selected denaturation curves from part b shows that for a given ph ph 7. Ligands are often used to increase the success rate of protein purification or crystallization by increasing the stability of the protein being studied. Sep, 2007 differential scanning fluorimetry dsf is a rapid and inexpensive screening method to identify lowmolecularweight ligands that bind and stabilize purified proteins. The thermofluor assay is a quick, temperaturebased assay to assess the stability of proteins. However, its applicability to membrane proteins is still limited because either large. Proteins are the key molecules studied as targets for new drugs as part of the lead generation process. Thermal shift and stability assays of diseaserelated. It can be used as an alternative to cd melts to determine melting temperatures.
The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. Pdf development of a thermofluor assay for stability. Mix protein, dye and dilution buffer thoroughly by inverting the tube several times and place the solution in a multichannel pipette reservoir trough. Transfer 10 l of each condition of a stability screen into the corresponding well of a 96well plate using a multichannel pipette to save time figure 4a. Thermofluor data analysis collaborative crystallisation. Apr 15, 2011 the thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence dsf, is a fast and simple method that is based upon high. The technology is simple, fast and insensitive to variations.
Figure 2 demonstrates the identification of a proteinbinding ligand, as shown by the increase in t. Current protocols in molecular biology edited by frederick m. Jan 01, 2015 a number of conditions are clearly detrimental to protein stability, such as c1, d2 and b12 bistris ph 6. We do the melts in triplicate, as dsf is a low information technique, and the redundancy helps us to trust the. The thermofluor method can be performed on nearly all qpcr machines, meaning no special instrumentation is necessary, and can be used to. The fluorescence dyebased differential scanning fluorimetry dsf method, also known as protein thermalshift assay or thermofluor, is a simple yet powerful tool that was initially developed to. The thermofluor screens jbscreen fundament and jbscreen specific allow identification of protein stabilizing buffer conditions which is pivotal for protein purification, characterization and crystallization. Protein thermal shift assay using applied biosystems real. If proteins of interest adopt nonnative congregating structures, normal function may be lost. From the thermal shift assay, a temperature at which the bulk of the protein becomes aggregated can readily be identified. Binding constants kb were measured by examining the systematic effect of ligand concentration on. Compatible with thermofluor and protein thermal shift assays.
The thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence dsf, is a fast and simple method that is based upon high. A systematic analysis of ph, buffer or salt identity and concentration, biological metals, surfactants, and common. Jan kostecki in steve mayos lab has been using this assay to assess the thermal stability of designed proteins. Thermofluor assay by jennifer keeffe april 20 background. The jbscreen thermofluor specific for protein stability a common reporter for protein stability is its melting temperature tm that can easily be determined by tsa thermal shift assay, fig. The aggregation temperature is an indicator of protein stability and. Pdf the stability and homogeneity of a protein sample is strongly influenced by the composition of the buffer that the protein is in. A general, rapid dyebinding thermal shift screen for protein engineering. Protein is destabilised by dtt, detergent, zn, fe, fad, tmp. Dsf is an excellent platform to screen for conditions that stabilize proteins, owing to the small amounts and low concentrations of protein required. For structural genomics, the bio logical function of the target proteins is often not known prior to the structural analysis. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins probability of yielding crystals, and optimization of these properties will improve success rates of crystallization.
The combined use of the thermofluor assay and thermoq analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Differential scanning fluorimetry dsf is a rapid and inexpensive screening method to identify lowmolecularweight ligands that bind and stabilize purified proteins. Thermofluorbased optimization strategy for the stabilization. Cs330 jbscreen thermofluor fundament jena bioscience. Conditions are optimized for differential scanning fluorimetry dsf assay to identify solution conditions which enhance protein stability and to optimize. Formulate the stability screens as 500 l aliquots in 96well blocks and seal for storage. Prepare 1 ml of an approximately 1 mg ml1 protein solution in an appropriate buffer system. The stability of therapeutic antibodies is a prime pharmaceutical concern. A collection and summary on thermofluor assay a corner. Md1103 the protein stability combo kit lubioscience. The jbs specific thermofluor screen uses the thermal shift assay methodology for identification of the specific variables for protein stability.
Oppf1446 protein is more stable in higher salt and ph. Thermofluorbased highthroughput stability optimization of proteins for structural studies. A common goal that many in vitro protein studies strive for is the creation of an environment in which protein samples can stably retain native conformations. We have developed a new method for measuring protein stability that can be used in high throughput screens. The use of differential scanning fluorimetry to detect ligand. May 29, 2016 protein stability protein stability is the net balance of forces, which determine whether a protein will be in its native folded conformation or a denatured state.
The method involves defining the thermal stability of a protein against a screen of buffer systems, systematically sampling ph from 5. Differential static light scattering dsls kurganov, 2002 differential scanning colorimetry circular dichroism nmr high throughput methods. Thermofluor based highthroughput stability optimization of proteins for structural studies. Thermofluorbased highthroughput stability optimization. Crystallization of membrane proteins is very laborious and timeconsuming, yielding well diffracting crystals in only a minority of projects. Thermofluor historically used to test protein stability. Glomelt thermal shift protein stability kit biotium. L of protein sample, in a concentration range from 0.
Thermofluorbased highthroughput stability optimization of proteins. Footandmouth disease fmd has a major economic impact throughout the world and is a considerable threat to food security. The combined use of the thermofluor assay and thermoq. We hope that this will be a useful alternative to existing methods for researchers looking for molecules that bind to. Thermal shift analysis of protein stability and ligand interactions. Optimization of protein buffer cocktails using thermofluor. Thermofluor, a miniaturized 384well thermal stability assay, has been implemented as a means of monitoring solutiondependent changes in protein stability, complementing the protein engineering and purification processes. At peak proteins, we determine the thermal melting temperature of our proteins as a routine quality control check. Methods to quantify capsid stability are currently limited, lack sensitivity and cannot accurately reflect differences in thermostability. Thermal denaturation assays in chemical biology assay and.
Proteinligand interactions investigated by thermal shift. An itd method to assess protein stability has also been optimized in a 384well format and has been reported to be very sensitive for the detection of small stability differences. The thermofluor assay has been developed as such a tool. The thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence dsf, is a fast and simple method that is based upon highthroughput measurements of protein stability.
Thermofluor based highthroughput stability optimization of proteins. The differing conditions that can be examined are very diverse, e. Thermofluorbased highthroughput stability optimization of. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies. In our standard dsf experiment buffer screen 10 conditions available on c6, we dilute your protein sample into a number of different buffers testing the stability of the protein at different ph and with two levels of salt.
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