otimização_thermofluor

Protein Expression and Purification 91 (2013) 192–206

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Protein Expression and Purification

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Review

Optimization of protein purification and characterization usingThermofluor screens

1046-5928/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.pep.2013.08.002

Abbreviations: ANS, Anilinonaphthalene-sulfonate; DSF, Differential Scanning Fluorimetry; DSC, Differential Scanning Calorimetry; TF, Thermofluor; RT-PCRtranscription polymerase chain reaction; Tm, Melting temperature.⇑ Corresponding author. Fax: +49 4089902149.

E-mail address: [email protected] (S. Boivin).

Stephane Boivin ⇑, Sandra Kozak, Rob MeijersEuropean Molecular Biology Laboratory (EMBL), Hamburg Outstation, Notkestrasse 85, 22603 Hamburg, Germany

a r t i c l e i n f o

Article history:Received 5 June 2013and in revised form 29 July 2013Available online 12 August 2013

Keywords:ThermofluorDifferential scanning fluorimetryFluorescence thermal shift assayThermodenaturationProtein stabilizationBuffer optimizationAdditive screenThermostabilityMelting pointCrystallization

a b s t r a c t

The efficient large scale production of recombinant proteins depends on the careful conditioning of theprotein as it is isolated and purified to homogeneity. Low protein stability leads to low purification yieldsas a result of protein degradation, precipitation and folding instability. It is often necessary to go throughseveral iterations of trial-and-error to optimize the homogeneity, stability and solubility of the proteinsample. We have set up Thermofluor assays to identify customized protocols for the preparation andcharacterization of individual protein constructs. We apply a two-step approach: we first screen for glo-bal parameters, followed by a search for protein-specific additives. The first screen has been designed insuch a way, that it is possible to discern global stability trends according to pH, salt concentration, buffertype and concentration. The second screen contains small molecules that can affect the folding, aggrega-tion state and solubility of the protein construct and also includes small molecules that specifically bindand stabilize proteins. The screens are designed to evaluate purification and storage protocols, and aim toprovide hints to optimize these protocols. The home-made screens have been tested on more than 200different protein constructs at the Sample Preparation and Characterization (SPC) facility at EMBL Ham-burg. We describe which RT-PCR machines can be adapted to perform Thermofluor assays, what are thenecessary experimental conditions to set up a screen, some leads on how to interpret the data and wegive several examples of Thermofluor applications beyond stability screens.

� 2013 Elsevier Inc. All rights reserved.

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193Thermofluor principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

RT-PCR machines compatible with Thermofluor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194Choosing the fluorophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194Preparing your sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Setting up the assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Performing the Thermofluor assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

High-throughput Thermofluor at the EMBL Hamburg facility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Buffer Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198Additive Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Data analysis and melting curve profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Plotting the melting curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202Single curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202Complex curves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Featureless curve. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203High initial fluorescence signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203Optimizing the assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
, Reverse

S. Boivin et al. / Protein Expression and Purification 91 (2013) 192–206 193

Thermofluor applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Screening the effect of mutations on stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203Measuring kinetics of ligand binding by Thermofluor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204Thermofluor and the crystallizability of protein samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204Thermofluor online resources. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Fig. 1. Typical thermal denaturation assay using Thermofluor. (A) Melting curve ofglucose isomerase in 100 mM Tris–HCl pH 8.5. The protein solution is heated in thepresence of a hydrophobic dye (SYPRO Orange). Upon denaturation, the dye bindsto the internal hydrophobic protein core increasing significantly the fluorescence(right Y axis). Maximal fluorescence intensity is obtained when the protein unfoldscompletely, then SYPRO Orange signal decreases corresponding to dye-proteindissociation. Residual signal from SYPRO Orange is explained by the interactionfrom the dye with aggregated protein. Usually the fluorescence signal is plotted as afunction of temperature to get a sigmoidal curve that shows the fraction of theunfolded protein (left Y axis). The inflection point corresponds to the meltingtemperature (Tm), at which 50% of the protein is unfolded. A Tm of 81.3 �C has beencalculated. (B) Alternative representation of the melting curve using the firstderivative – (dRFU)/dT of the raw data: the Tm corresponds to the apex. (Forinterpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

Introduction

The characterization of individual proteins often requires thatthey are produced in quantities that go far beyond their abundancein the cell. They are isolated in an environment that is devoid ofpotentially stabilizing factors. It is not surprising that many proteinsamples show loss of function and reduced stability in standardsample buffer conditions. It is therefore useful to identify the com-ponents that are essential to recover the integrity and activity ofthe protein, and this identification process itself can contribute toan understanding of the protein’s function. The necessary activa-tion ingredients can often be inferred from prior knowledge ofthe protein species, but some may be unknown. Here, we presentan approach to screen common environmental factors such aspH, buffer type, ionic strength and a range of crowding agentsusing Thermofluor.

Recombinant protein expression often results in large quanti-ties of the protein of choice that will yield sufficient amounts forcharacterization through brute force purification. The resultingprotein sample may seem pure at face value, but it is importantto check its quality in terms of protein activity, integrity, dispersityand stability. Structural studies are especially demanding, becausethe protein sample has to remain stable under excruciating exper-imental conditions. Crystallization requires highly concentratedsamples and can take any time from several hours to weeks. Stan-dard protein crystallization is done at room temperature, andmany proteins are not stable over long time periods under theseconditions. Thermostability is therefore an important feature thatneeds to be tested, and protein constructs may have to be opti-mized with stability in mind.

A common approach is to use protein homologues fromextremophiles, i.e., species that live in circumstances of extremetemperature and pressure. These proteins have evolved their struc-ture so they can still function under extreme conditions [1], andthey are more stable under standard crystallization conditions.Classical examples for the crystallization of proteins from thermo-philic organisms are the 70S ribosome [2] and the potassium ionchannels [3]. When switching species is not an option, it is possibleto optimize the protein construct though truncations, mutations orfusions with stabilizing factors. Proteins can be co-expressed withother partners that help to stabilize the protein fold. In the end, theprotein sample can be further stabilized by adjusting pH, salt con-centration, buffer system or by adding additives. The identificationof a stabilizing solution increases the ability to purify, concentrateand crystallize the protein [4,5]. Unfortunately, no clear correlationhas been observed between stability and intrinsic properties of aprotein such as the isoelectric point, molecular weight and per-centage of charged residues. No empirical rules can be drawn;therefore we strongly recommend measuring the thermal stabilityof each new recombinant protein and systematically screen foroptimum conditions.

There are a number of techniques that can be used to monitorthe effect of external factors on the thermostability of the sample.

194 S. Boivin et al. / Protein Expression and Purification 91 (2013) 192–206

Circular dichroism coupled to a temperature gradient can be usedto monitor the loss of secondary structure as a sign of the unfoldingof the protein at elevated temperatures [6]. Protein aggregationcoupled to a temperature gradient can be monitored by light scat-tering based methods [7,8] or by measuring the optical density ofthe sample [9]. Differential scanning calorimetry (DSC)1 is anothernon-evasive technique to study protein thermostability [10], but it isvery time consuming. Here, we discuss the adaption of a techniquethat was developed by the pharmaceutical industry to screen for li-gands that thermally stabilize protein targets. Thermofluor has beenshown to be useful to assess thermostability in a systematic way,and it enables many conditions to be tested simultaneously. Thetechnique requires relatively small amounts of sample and is inex-pensive. Many structural genomic consortia use Thermofluor as astandard technique for quality control and to optimize sample con-ditioning. However, anyone who has access to a real time PCR ma-chine can modify it to screen protein thermostability.

Thermofluor principle

The fluorescence-based thermal stability assay or differentialscanning fluorimetry (DSF) has been developed by Pantolianoand coworkers in 2001 [11]. Registered as a trademark of 3-Dimen-sional Pharmaceuticals (3DP), the term Thermofluor subsequentlypassed into common usage [12]. The methodology takes advantageof the fact that the fluorescence of many nonspecific protein-bind-ing dyes increases with increasing hydrophobicity of their environ-ment [13,14]. In the procedure, a dye interacts with exposedhydrophobic regions generated by partial or full unfolding of pro-teins. It is based on the detection of changes in the exposure of aprotein’s hydrophobic core upon heat denaturation. The dye isquenched in aqueous solutions, but when the aromatic moietiesof the dye intercalates into a hydrophobic pocket, it regains itsfluorescence. For many proteins, the gradual increase of tempera-ture has little effect on the protein fold, until a temperature isreached where it will quickly unfold. At this point, the unfoldedprotein will expose its hydrophobic core and the dye will becomefluorescent (Fig. 1A). In an ideal case, the sudden unfolding of allthe proteins in the sample will lead to a sharp increase of the fluo-rescent signal over a short temperature range. A sharp sigmoidalcurve allows for the calculation of a melting temperature (Tm),which correspond to the temperature where the protein is 50% un-folded. The melting temperature can also be derived by calculatingthe peak of the first derivative (Fig. 1B). It has been shown that asharp melting curve is highly reproducible, and this opens the pos-sibility to compare melting temperatures between samples thatcontain the same protein but different buffer compositions. A posi-tive shift in the melting temperature DTm can be coupled to an in-crease in structural order and a reduced conformational flexibility,whereas a negative DTm, indicates that the buffer induces proteinstructural changes towards a more disordered conformation.Although this process can in principle be observed with any fluo-rimeter with a thermoblock, the most ideal configuration is ob-tained with a real-time PCR machine. These thermocyclers areequipped to detect fluorescence in multi-well plates so that manyconditions can be tested simultaneously.

RT-PCR machines compatible with Thermofluor

The adaption of commercially available RT-PCR machines forThermofluor has made this technique easily accessible [15]. In

1 Abbreviations used: ANS, Anilinonaphthalene-sulfonate; DSF, Differential ScanningFluorimetry; DSC, Differential Scanning Calorimetry; TF, Thermofluor; RT-PCR,Reverse transcription polymerase chain reaction; Tm, Melting temperature.

principle, any RT-PCR instrument can be used for a Thermofluorassay as long as the optical system of the instrument is compatiblewith the fluorescent properties of the dye used in the assay. Instru-ments reported to have been used for thermal shift assays includethe Mx3005P (Stratagene) and the iCycler (Bio-Rad) for a 96-wellformat assay, the LightCycler 480 (Roche) and the FluoDia T70(PTI) for a 384-well format assay, while the ABI 7900 (AppliedBiosystems) provides both 96- and 384-well format assays. Thechoice of the instrument depends mainly on the required capacity,the type of dye, the fluorescent filters available, and the tempera-ture range required. An RT-PCR machine equipped with a coolingsystem provides the advantage to analyze protein stability from4 �C upwards, which can be crucial for temperature sensitive pro-teins. At the SPC facility, we use an iCycleMyIQ RT-PCR DetectionSystem (Bio-Rad), equipped with a charge-coupled device (CCD)detector for imaging of the fluorescence. This RT-PCR is designedto support the use of a single filter pair optimized for excitationand emission of green fluorescent dyes, resulting in excellent sen-sitivity for the detection of fluorophores such as FAM and SYBRGreen I (with excitation maxima at 485 and emission spectra at530 nm, respectively). These filters have shown to be compatibleand useful to perform thermal denaturation of proteins using SY-PRO Orange which, upon binding protein, has a fluorescence exci-tation/emission maximum spectra at 470 and 569 nm. However,filters can also be replaced manually and adaptations can be madefor dyes with different fluorescent properties. If required, custom-ized filters can be purchased from CHROMA technology (http://www.chroma.com).

Choosing the fluorophore

Various fluorescent dyes have been tested to probe changes inprotein conformation [16]. Nile red is a fluorescent probe for intra-cellular lipids and hydrophobic protein domains with a maximumabsorption at 553 nm. The 1,8-ANS dye makes hydrophobic andelectrostatic interactions with proteins with a maximum absorp-tion at 350 nm. The bis-ANS dye binds stronger to the hydrophobiccavities of the protein than 1,8-ANS, and has a maximum absorp-tion at 385 nm. However, the most popular dye for thermal shiftassays is SYPRO Orange [17–19].

Fig. 2. Effect of a His6-tag on the thermal stability of a viral protein. Thermalstability of the pure protein was measured using Thermofluor before and aftercleavage of the purification tag using TEV protease. The untagged protein shows athermal shift of 4.1 �C compared to the tagged protein. This illustrates that it isimportant to verify the effect of protein purification tags on protein stability.


The SYPRO Orange dye is relatively sensitive with a high signalto noise ratio for the fluorescence upon protein binding [17,20].The fluorescent properties of the dye-protein complex with a max-imal absorption at 470 nm and maximal emission at 569 nm [17],minimize the interference with background fluorescence fromsmall molecules. For all environmentally sensitive dyes includingSYPRO Orange, the Thermofluor effect depends on the partitioningbetween the high-dielectric environment of water and the ‘‘hydro-phobic’’, low-dielectric environment that occurs when a protein ismelted. In some cases, interaction with native proteins, e.g., viahydrophobic pockets on the protein surface, as well as interactionwith detergent leads to a high initial signal background. For thisreason, systematic efforts to understand membrane protein stabil-ity with Thermofluor assays is challenging. There are alternativedyes such as the thiol-specific fluorochrome N-[4(7-diethyl-amino-4-methyl-3-coumarinyl]maleimide (CPM) which reactswith cysteines that become accessible when a protein unfolds[21]. To overcome this problem Enzo Life Sciences developed theProteostat� dye, which is compatible with detergents and whichmonitors protein aggregation rather than protein unfolding[22,23]. If the protein is not compatible with any of the fluorescentdyes available, Warne and coworkers reported the development ofa dye-free microscale assay to measure the thermal stability of amembrane protein depending on 3H labeled ligand binding [24].

Preparing your sample

In order to obtain a reliable Tm value, it is recommended to usehighly purified protein for the assay. It is occasionally possible toobtain a sharp melting curve with proteins that are 60% pure, butwe generally recommend a purity of 75% or higher. Special cautionshould be taken with recombinant and over-expressed proteins asthey often contain modifications, such as the addition of affinity orsolubility tags. A solubility tag can add features to the meltingcurve that are hard to distinguish from the protein of interest. Inthe worst case, it can give the completely misleading impressionthat the protein is folded, whereas the tag is the only part of therecombinant protein that is folded and contributing to the meltingcurve. On the other hand, an affinity tag such as a 6�His-tag orstreptavidin-tag can decrease the protein thermal stability. Weshow an example in (Fig. 2), where the cleavage of a C-terminal6xHis-tag with TEV protease leads to an increase in the meltingtemperature of 4.1 �C. It should be kept in mind that the peptideaffinity tags have a highly dynamic structure and can initiate thedestabilization of the tagged protein. It is generally desirable to re-move tags, and ensure that other modifications have not influ-enced protein folding or integrity.

The initial protein sample should not contain high concentra-tions of buffers or elution agents such as imidazole, because thesemay obviously interfere with the final pH of the assay. Since thesample in our described setup is diluted in the Thermofluor bufferby a factor of 10, we recommend to prepare the sample in a lowionic strength environment; with salt and buffer concentrationsno higher than 200 mM at a neutral pH. It is recommended touse a sample buffer free of stabilizing reagents such as glycerol,reducing reagents such as DTT or TCEP and detergents. Thesechemicals can influence protein stability or may interfere at highconcentration with the fluorescence measurement. If the use ofstabilizing agents cannot be avoided, it is advised to modify theirconcentrations in separate assays to assess their influence.

Setting up the assay

A typical thermal stability assay will require for each conditiontested an aliquot of 2 ll of purified-protein at an initial concentra-tion of �20 lM. A typical assay volume is 25 ll per well so the final

protein concentration will be 1.6 lM. For a 96-well assay, 115 lgof a protein with a molecular weight of 30 kDa will be required.The dye signal is concentration dependent; a higher protein con-centration will result in a higher signal to noise ratio but may affectthe oligomerization state of the protein and cause a higher back-ground signal. Under favorable circumstances, it is possible tomeasure a melting curve with a protein concentration lower than5 lM. However, it should be kept in mind that small proteins(<15 kDa) tend to generate a blurry transition shape that may behard to extract from a weak signal. In case a protein cannot be con-centrated to 20 lM, we suggest using a larger volume of the pro-tein sample for the assay in order to reach a final concentrationof 1.6 lM. The mixing protocol should then be modified to keepa final volume of 25 ll. The use of a larger volume of the proteinsample solution can influence the final chemical environment ofthe assay, and this should be taken into consideration.

SYPRO Orange (Invitrogen) is delivered as a 5000� concen-trated solution in DMSO. For a 96-well assay, we prepare a solutionof SYPRO Orange by diluting 3 ll of the 5000� concentrated dyeinto 237 ll of distilled water to have a working solution that is62� concentrated. For each condition, 2 ll of 62� SYPRO Orangeis needed per well to reach a final concentration of 5�, which isthe typical concentration used in most experiments. At this dyeconcentration, the fluorescent response is practically independentof the size of the protein.

The mixing protocol we use is in the following order: (1) Suffi-cient distilled water to reach a total volume of 25 ll, (2) 5 ll of 5�buffer, (3) 5 ll of 5� salt [optional], (4) 5 ll of 5� additive [op-tional], (5) 2 ll of 20 lM protein and then 2 ll of SYPRO Orangesolution. We advise against pre-mixing the protein and the dye, be-cause the dye contains DMSO. This solvent can damage the proteinin higher concentrations, or it can interact with the protein affectingthe initial background signal during the Thermofluor experiment.The PCR-plate should be kept on ice during the preparation in orderto prevent protein denaturation and to equilibrate the sample forthe starting temperature of the assay.

Performing the Thermofluor assay

Once the microplate has been filled with samples and buffers,the plate is sealed with highly transparent optical-clear qualitysealing tape (e.g., Greiner Bio-one, catalogue number 676070).The plate is centrifuged at 4 �C at 2500g for 30 s immediately be-fore the start of the assay to remove possible air bubbles. TheRT-PCR machine can be programmed first with a 5 min equilibra-tion time at 5 �C to allow SYPRO Orange to diffuse. We have ob-served that temperature equilibration of the assay lowers theinitial background fluorescence. Subsequently, the plate is heatedfrom 5 to 95 �C (20 to 95 �C if no cooling system is available) withinitial stepwise increments of 1 �C per minute, followed by thefluorescence reading optimized for SYPRO Orange at 485/20 nm(Ex) and 530/30 nm (Em). When it is needed to record the meltingtemperature at higher precision, smaller temperature incrementscan be used. It has been reported that the heating gradient can af-fect the precision of the Tm within the experimental error (Tm -� 0.2 �C), but that it will not affect the DTm measured betweentwo experimental conditions [15]. The entire procedure requiresless than two hours. We present a summary of the procedure in or-der to set up a high-throughput thermal stability assay (Fig. 3).

High-throughput Thermofluor at the EMBL Hamburg facility

Samples that enter the SPC facility for optimization are firstgoing through a quality control protocol that includes a singleThermofluor experiment. In this single experiment the buffer

Fig. 3. Overview of the protocol for a high-throughput Thermofluor assay. First, all stock solutions need to be prepared in a 96 deep well block; buffer (5� concentrated), salt(5� concentrated) and water. A stock solution of additives (5� concentrated) can be also prepared when screening for further stabilization. These deep well blocks with stocksolutions can be stored at �20 �C, if its chemical contents allow. Afterwards, to prepare the 96-microplate, 5 ll of each component is added to the plates, and then the volumeadjusted to 21 ll with distilled water. When preparing the 96-microplates in batch, we recommended to use a liquid handling robot system or a multichannel pipette tooptimize time and assure reproducibility between experiments. If the plates are not used the same day, they need to be sealed immediately to prevent evaporation and storedat �20 �C. When retrieved from storage, 96-microplates need to be thawed at room temperature and centrifuged (4 �C, 30 s, 2500g). A working solution of SYPRO Orange 62�need to be prepared freshly and added to each well of the 96-microplate to reach a final concentration �5� per well; by diluting 3 ll of SYPRO Orange 5000� to 237 ll ofwater. Then 2 ll of protein and 2 ll of dye-working solution is added with a repeater pipette to each of the 96 wells. Final volume per well is 25 ll. The PCR plate is sealedwith a optical clear lid, centrifuged (4 �C, 30 s, 2500g) and is being analysed on a real-time PCR machine using a temperature gradient of 1 �C/min from 5 to 95 �C. The wholeassay lasts approximately 2 h. The melting curves from each individual well have to be processed with appropriate software. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)


consists of 100 mM Hepes pH 7.4 and 100 mM NaCl. This buffer isin general suitable for a preliminary test for a large number of pro-teins. Based on the initial assessment of the Tm value and profile ofthe melting curve, we advise our facility users to follow a stepwiseapproach to find the most optimal sample conditions. We first tryto assess the effect of global parameters including buffer type andconcentration, pH and salt concentration in a systematic way witha ‘‘Buffer Screen’’ using 96 different conditions (Table 1). This screenis suitable for proteins for which globally optimal conditions haveto be identified. The screen is set up so that better handling condi-tions can be designed for the protein, for instance during proteinpurification. The identification of optimal conditions is useful forproteins that cannot be frozen without loss of activity and needto be stored at 4 �C for a long time. The screen has also been usefulto optimize the conditions for the study of protein–protein interac-tions where the proteins are first screened individually and then

mixed together to assess if this changes the behavior of the proteincomplex in the ‘‘Buffer Screen’’ assay. Once the most stabilizingglobal parameters have been established, the protein sample isconditioned accordingly. In a second iteration process, the ‘‘Addi-tive Screen’’ can be used to find additional factors that stabilizethe protein, favor crystallizability, or may hint at potential proteinligands and even functional aspects (Table 2). We have found thatfor almost all proteins investigated in our facility with thermalshift methodology, conditions were found that were more stabiliz-ing than the starting buffers condition. Below, we present thescreens we developed in detail and the rationale for its compo-nents. When the screens are run in the absence of protein sample,the components themselves do not have a fluorescent fingerprint.In the presence of protein sample, some components increase thefluorescent background, without affecting the overall interpreta-tion of the thermal shifts.

Table 1Layout of the Thermofluor Buffer Screen.

1 2 3 4 5 6 7 8 9 10 11 12

A Water Citric Acid Na Acetate Citric Acid MES KH2PO4 Citric Acid Bis-Tris Na Cacodylate NaH2PO4 KH2PO4 HEPESpH 4.0 pH 4.5 pH 5.0 pH 6.0 pH 6.0 pH 6.0 pH 6.5 pH 6.5 pH 7.0 pH 7.0 pH 7.0

B MOPS Am Acetate Tris–HCl NaH2PO4 Imidazol HEPES Tris–HCl Tricine Bicine Bicine Tris–HCl BicinepH 7.0 pH 7.3 pH 7.5 pH 7.5 pH 8.0 pH 8.0 pH 8.0 pH 8.0 pH 8.0 pH 8.5 pH 8.5 pH 9.0

C Water Citric Acid Na Acetate Citric Acid MES KH2PO4 Citric Acid Bis-Tris Na Cacodylate NaH2PO4 KH2PO4 HEPESNaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl

pH 4.0 pH 4.5 pH 5.0 pH 6.0 pH 6.0 pH 6.0 pH 6.5 pH 6.5 pH 7.0 pH 7.0 pH 7.0

D MOPS Am Acetate Tris–HCl NaH2PO4 Imidazol HEPES Tris–HCl Tricine Bicine Bicine Tris–HCl BicineNaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaClpH 7.0 pH 7.3 pH 7.5 pH 7.6 pH 8.0 pH 8.0 pH 8.0 pH 8.0 pH 8.0 pH 8.5 pH 8.5 pH 9.0

E Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’ Buffer ‘‘A’’pH 4.0 pH 4.78 pH 5.21 pH 5.62 pH 5.95 pH 6.23 pH 6.53 pH 6.81 pH 7.16 pH 7.80 pH 9.0 pH 10.0

F Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’ Buffer ‘‘B’’pH 4.0 pH 4.22 pH 4.62 pH 5.06 pH 5.62 pH 6.49 pH 7.25 pH 7.76 pH 8.20 pH 8.75 pH 9.0 pH 10.0

G 10 mM HEPES 50 mM HEPES 100 mM HEPES 250 mM HEPES 10 mM 50 mM 100 mM NaPO4 200 mM NaPO4 10 mM Tris–HCl 50 mM Tris–HCl 100 mM Tris–HCl 250 mM Tris–HClpH 7.5 pH 7.5 pH 7.5 pH 7.5 NaPO4 NaPO4 pH 7.5 pH 7.5 pH 8.0 pH 8.0 pH 8.0 pH 8.0

pH 7.5 pH 7.5

H 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM 50 mMHEPES HEPES HEPES HEPES HEPES HEPES Tris–HCl Tris–HCl Tris–HCl Tris–HCl Tris–HCl Tris–HCl50 mM NaCl 125 mM NaCl 250 mM NaCl 500 mM NaCl 750 mM NaCl 1 M NaCl 50 mM NaCl 125 mM NaCl 25 mM NaCl 500 mM NaCl 750 mM NaCl 1 M NaClpH 7.5 pH 7.5 pH 7.5 pH 7.5 pH 7.5 pH 7.5 pH 8.0 pH 8.0 pH 8.0 pH 8.0 pH 8.0 pH 8.0

Buffers were used at concentration of 100 mM unless otherwise indicated. Sodium chloride was used at concentration of 250 mM, unless otherwise indicated. Buffer ‘‘A’’ composition: Succinic Acid/NaPO4/Glycine [2:7:7]; Buffer ‘‘B’’ composition: Citric Acid/CHES/HEPES [2:4:3].

Table 2Layout of the Thermofluor Additive Screen.

1 2 3 4 5 6 7 8 9 10 11 12

A 0.1 M 0.5 M 1 M 2 M 4 M 6 M 150 mM 500 mM 3% (v/v) 100 mM 100 mM 100 mMUrea Urea Urea Urea Urea Urea GdnHCl GdnHCl DMSO NaHCO2 KHCO2 NH4HCO2

B 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mMNaC2H3O2 Ca(C2H3O2)2 CH3CO2K CH3COONH Na2SO4 Mg2SO4 K2SO4 (NH4)2SO4 Na2HPO4 NaH2PO4 K2HPO4 KH2PO4

C 100 mM 100 mM 100 mM 100 mM 10 mM 1 mM 5 mM 20 mM 100 mM 1 mM 1 mM 5 mMNa2C4H4O Na3C6H5O7 Na2C3H2O4 NaNO3 DTT TCEP TCEP TCEP TMA-HCl Spermidine Spermine-HCl EDTA

D 10 mM 50 mM 100 mM 250 mM 500 mM 10 mM 10 mM 1 mM 1 mM 1 mM 1 mM 1 mMBetaine Imidazole Imidazole Imidazole Imidazole MgCl2 CaCl2 MnCl2 NiCl2 FeCl3 ZnCl2 CoCl2

E 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 2 mM 1 mMNaF KF NH4F LiCl NaCl KCl NH4Cl NaI KI NaBr CHAPS OG

F 3% (v/v) 1% (v/v) 5% (v/v) 10% (v/v) 20% (v/v) 5% (v/v) 5% (w/v) 5% (w/v) 10 mM 50 mM 50 mM 25 mMEG Glycerol Glycerol Glycerol Glycerol PEG 400 PEG 1000 PEG 4000 Pro Taurine Gly His

G 50 mM 50 mM (each) 500 mM (each) 50 mM 25 mM 50 mM 100 mM 100 mM 100 mM 100 mM 2 mM 2 mMArg Arg + Glu Arg + Glu Glu Gln Lys D-Glucose Sucrose Maltose D-Sorbitol NADH ATP

(5 mM MgCl2) (5 mM MgCl2)

H 2 mM 2 mM 2 mM 2 mM 2 mM Buffer Custom Custom Custom Custom Custom CustomADP cAMP GTP GDP cGMP(5 mM MgCl2) (5 mM MgCl2) (5 mM MgCl2) (5 mM MgCl2) (5 mM MgCl2)

Abbreviations: guanidine hydr Chloride (GdnHCl), trimethylamine (TMA), Tris(2-carboxyethyl)phosphine (TCEP), ethylenediaminetetraacetic acid (EDTA), ethylene glycol (EG), Ctyl-b-D-glucoside (OG). The additive screen contains: chaotropic/dissociationreagents [A1:A9], salts [A10:C4], reducing reagents [C5:C8], polyamines [C9:C11], chelating agent [C12], linker [D1], imidazole [D2:D5], multivalent ions [D6:D12], monovalent ions [E1:E10], detergents [E11:E12], polyols [F1:F8], amino acids [F9:G6],carbohydrates [G7:G10], co-factors [G11:H5].

S.Boivinet

al./ProteinExpression

andPurification

91(2013)

192–206

197

Fig. 4. Effect of buffer and pH on glucose isomerase thermal stability. Representative melting curve from different buffers over pH 6–9. The results obtained in absence ofbuffer are indicated as a black line and those in presence of buffers in colored lines. The different melting points extracted from the melting curves for the more stabilizing anddestabilizing conditions are given in the insert. Compared to water (Tm of 76.1 �C), Tris–HCl pH 8.0 (blue) is shown to be the most stabilizing condition increasing the Tm byD + 5.9 �C (82.0 �C) whereas MES buffer at pH 6.0 is shown to be the most destabilizing condition with a Tm of 71.5 �C. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

Fig. 5. Effect of pH on glucose isomerase thermal stability. Representative meltingcurve at pH 4–10 using an extended-range buffer system composed of succinic acid/NaH2PO4/glycine (2:7:7). The aim of such a buffer system is to deconvolute theeffect of pH from the buffer himself. In this example, pH’s below 6.2 show a strongdestabilizing effect while the optimal pH range is 6.5–9.0. The melting temperaturefor the three component buffer system is lower for the whole pH range than what isobtained for Tris buffer. The pH effect shows globally that it is preferred to workwith neutral or basic pH, but the choice of the actual buffer system is crucial.


Buffer Screen

Choosing the right buffer system for a macromolecular sampleis vital from the first extraction through purification and character-ization of the protein. In some cases, buffers such as Tris have beenreported as a source of sample heterogeneity [25]. In contrast,buffer systems can have a positive effect on the protein stabilityby modulating the pH of the protein solution and by directly inter-acting with the protein itself. It is common to find ordered buffermolecules within protein crystal structures that form intrinsicinteractions within the protein. To date, there are more than 600structures in the Protein Data Bank [26] that contain orderedMES, about 500 that contain ordered Hepes, and more than 600that contain ordered Tris, showing that it is not unusual to havespecific interactions between buffers and protein molecules. Forexample, one of the structures of glucose isomerase reports thepresence of a Tris molecule (PDB ID 1MNZ). It is therefore essentialto test the effect of different buffer systems on protein stability.

This section describes our ‘‘Buffer Screen’’ which contains a set of23 common buffer systems (16 different chemicals) each at a

concentration of 100 mM covering a pH range from 4.0 to 9.0 inabsence or in presence of 250 mM NaCl to favor protein solubility(Table 1, conditions A1–B12 and C1–D12, respectively). Weincluded a condition with water only as a reference control toevaluate the effect of the absence of a buffer system on the meltingtemperature. Our aim is to assess whether a protein showsincreased stability for a certain buffer system over a certain pHrange. As a case study, we present the outcome of the ‘‘BufferScreen’’ on glucose isomerase (Fig. 4). An issue in optimization isto find factors which are independent, so as to simplify the analysisof the results of the optimization trials. This can be a challengewith buffers, as a buffer and its pH range tend to be highly corre-lated. Moreover, some buffer properties are affected by otherfactors. For instance, the buffer capacity of Tris is temperature-dependent. Our ‘‘Buffer Screen’’ was designed in such a way thatthese trends can be deconvoluted (Fig. 5). Since most single buffersystems have a narrow pH range, we suggest to test at least oneextended-range buffer system as these systems ensure a smoothgradient in pH and a consistent buffering capacity without neces-sitating a change in the buffer components. A multi-buffer systemfor pH modulation is useful to identify the best stabilizing pH usinga fine grid by titrating surface groups. The ratio of the componentswithin the buffer system are selected so as to produce a reasonablylinear response to pH [27]. We included in our ‘‘Buffer Screen’’ thesuccinic acid/phosphate/glycine buffer as well as the citric acid/Hepes/Ches buffer system to cover a pH from 4 to 10 (Table 1, con-ditions E1–F12). In most cases, we obtained a very good correlationbetween results from these two buffers systems within 0.3 pHunits and we can identify specific buffer effects. A similar strategyhas been used successfully to assess the variation in stability of theCorA Mg2+ transport channel with pH [28]. In addition, some affin-ity purification protocols involve a pH shock to elute the proteinwhich may not be suitable for the folded protein as it results inprotein denaturation, precipitation or aggregation [29,30]. Basedon the trends observed in the ‘‘Buffer Screen’’, it can be useful tomodify the sample purification protocol. The nature of the bufferand the pH are not the only important parameters to optimize withthe aim to increase protein stability. Given the potential interac-tions between certain buffers and proteins, it is also important togauge the effect of buffer concentration on protein stability. The‘‘Buffer Screen’’ samples the effect of the buffer concentration(10 mM up to 250 mM) for three buffers widely used for proteinpurification and characterization; Hepes, phosphate and Tris

Fig. 6. Effect of ionic strength on a protein’s thermal stability. Representative melting curves of an (A) adaptor protein at different concentrations of NaH2PO4 from 10 to200 mM and (B) DNA binding domain in presence of NaCl from 0.05 M to 1 M in 50 mM Tris–HCl pH 7.5. In both cases, the calculated Tm value increases with the ionicstrength of the solution. In the case of DNA binding domain, the addition of 1 M NaCl increases the Tm value by +7.8 �C. This data has been used to titrate the optimal NaClconcentration for NMR studies, and it was shown that 225 mM NaCl promotes protein stability without having an impact on DNA binding.


(Table 1, conditions G1–G12). We provide an example whereprotein thermostability of an adaptor protein is dependent on buf-fer concentration, increasing the Tm up to D + 10.8 �C (Fig. 6A).These results indicate that the ionic strength might be a key factoraffecting the thermostability of proteins [31].

Protein stability is also strongly affected by salt ions incorpo-rated in the hydration shell. Too high or too low salt concentrationscan lead to precipitation of a protein. For this reason, we imple-mented a NaCl concentration screen (Table 1, condition H1–H12)to further assess how ionic strength affects protein stability. Titra-tion for the optimal NaCl concentration has shown in some cases tohave a strong stabilizing effect and may contribute to find opti-mum conditions for structural studies. For example, a DNA bindingdomain of Sfc1 (from Schizosaccharomyces pombe) was not behav-ing well in its standard buffer during NMR measurements. Thethermostability of this protein was tested by Thermofluor usingthe ‘‘Buffer Screen’’, which helped to identify a strong stabilizing -effect induced by NaCl. Fine-tuning of the experimental conditionsallowed to determine the concentration of NaCl suitable to pro-mote the protein stability in solution (up to 225 mM NaCl) withoutimpairing affinity to DNA (Fig. 6B). While ionic strength plays a keyrole in the stability of several proteins, no clear trend has beenobserved between the stabilizing effect of buffer and NaCl concen-tration, suggesting that both of these parameters should be opti-mized independently. In summary, the ‘‘Buffer Screen’’ designed

for our facility explores the effect of four basic parameters on theprotein thermal stability (buffers, pH, buffer and NaCl concentra-tion) to identify an optimal buffer environment.

Additive Screen

Ligand-induced conformational stabilization of proteins is awell-understood phenomenon. Substrates, inhibitors, cofactors,and protein binding partners provide enhanced stability to pro-teins by selective binding. A thermal denaturation assay can beused to screen for the effect of additives while the buffer condi-tions are kept constant. Upon ligand binding, the protein complexdenaturates at a higher temperature and the difference in the Tm

value in the presence and absence of the compound reflects ligandbinding. Thus, the thermal shift assay can serve as a tool to seek forstabilizing reagents, and to identify natural ligands that provide in-sight into the biological function of the protein [20,32]. The ‘‘Addi-tive Screen’’ requires highly purified and active protein, to ensurethat the additive-protein interaction is the sole cause of the in-crease in stability. Once a ligand has been identified, it is importantto include it in the early stages of protein production, sometimesalready during protein expression to promote proper folding andto prevent aggregation. Many proteins or domains remain unstableor partially soluble in absence of their cofactors or stabilizingligands [4].

Fig. 7. Effect of imidazole concentration on the thermal stability of a protein involved in the immune system. Imidazole is widely used as an elution buffer to remove His-tagged recombinant proteins from a metal–chelate resin. Here we show that imidazole can have a strong destabilizing effect on proteins, leading to aggregation and proteinprecipitation. The concentration of imidazole should be adjusted in order to minimize the destabilizing effect on the folding of the protein which can occur at imidazoleconcentrations as low as 50 mM. The standard elution protocol for this protein used 250 mM imidazole, which destabilizes this particular protein with a thermal shift of�15.3 �C.


At the SPC facility, once we identified a suitable buffer using theinitial ‘‘Buffer Screen’’, the proteins are rescreened with the newoptimal buffer system against the ‘‘Additive Screen’’. The ‘‘AdditiveScreen’’ consists of a selection of 72 different physiological andnon-physiological ligands that include amino acids, nucleotides,sugars, cofactors, monovalent and divalent ions, and some otheradditives representing 14 categories (Table 2). We typically select4–10 commercially available compounds per framework. It is con-venient to prepare stocks of 5� concentrations of all compoundsand dispense 5 ll into each well (Fig. 3). The final concentrationsof the compounds within the screens varies between 1 mM and100 mM, depending on their solubility limit. In principle, a com-pound can show an effect at 10 lM concentrations when the bind-ing affinity is expected to be between 1 nM and 1 lM. However,compounds that bind with relatively low affinity such as nucleo-tides have to be present in high concentrations. The ‘‘AdditiveScreen’’ is not exhaustive, and it is advised to customize it basedon prior knowledge of the protein’s function. We designed thisscreen especially to further optimize protein purification and favoroverall protein stability. Artificial and natural stabilizing com-pounds as well as chaotropic reagents are included since they havethe potential to prevent protein aggregation.

We have shown with the ‘‘Buffer Screen’’ that the nature of thebuffer and the ionic strength can have an important effect on pro-tein stability. The presence of ions might induce changes of proteinsolubility, protein denaturation and changes in enzyme kinetics bymodulating electrostatic interactions within the protein. We in-cluded 17 ion species in the ‘‘Additive Screen’’, varying the combina-tion of cations and anions (Table 2, conditions A10–C4, E1–E10)and metals ions (Table 2, conditions D6–E10) which are widelyused for protein purification and characterization. For example,using this screen it is possible to compare the stability of the pro-tein with different halogens, to see if the commonly used NaCl hasa deleterious effect when replaced with NaF. Moreover, since NaClis not compatible with circular dichroism measurements in the far-UV range, NaF is an interesting alternative.

Divalent ions and cations may coordinate with the protein andact as generic stabilizers [31]. For example, the addition of MgCl2 toribonuclease A [33] and the CorA Mg2+ transport channel from Met-hanococcus jannaschii showed a positive thermal shift [28]. Diasand coworkers performed a Thermofluor assay on the PA subunitof influenza virus polymerase [34] and observed a substantial shiftfor divalent metal ions (exceeding 10 �C). Based on this assay, theyshowed that this domain has intrinsic RNA and DNA endonucleaseactivity that is strongly activated by manganese ions, matching

observations reported for the endonuclease activity of the intacttrimeric polymerase. We encourage to gauge metal affinity usingdifferent concentrations of metal ions, since proteins can havemultiple metal binding sites. However, caution should be takensince it has been reported that metal ions such as FeCl2, CuCl2,and CoCl2 have a tendency to quench fluorescence in thermal sta-bility assays at high concentration (mM) [35]. In our ‘‘AdditiveScreen’’, this effect was minimized for 7 metals by using low con-centrations (1–10 mM) (Table 2, conditions D6–D12). Given theside effects, it is advisable to further test the optimal concentrationof metal ions once a metal dependency has been observed. We alsosuggest to use fresh metal preparations that are no more than afew weeks old, since compounds such as FeCl2 tend to oxidize overtime.

The most common first purification step for proteins preparedfor structural characterization is affinity chromatography basedon the binding of a poly-Histidine tag to beads charged with Nickelor Cobalt. Imidazole is commonly used to elute the recombinantproteins from the affinity column. This procedure involves a highconcentration of imidazole (250–500 mM) which may affect pro-tein stability. Many proteins precipitate immediately when theimidazole is dialyzed out. The combination of a suboptimal buffercondition and the presence of high concentrations of His-taggedprotein can promote protein aggregation and precipitation. For thisreason, it is useful to explore the stability of the protein in differentconcentrations of imidazole to identify the highest concentrationwhere it does not affect protein stability. This upper concentrationcan then be used in the elution buffer. We have included an imid-azole gradient in the ‘‘Additive Screen’’ from 10 mM up to 500 mM(Table 2, conditions D2–D5), see example (Fig. 7). This exampleshows a strong destabilizing effect even at low imidazole concen-trations. In such cases, we recommend to use pH shock as an elu-tion method instead of an imidazole gradient. If this does not work,it may be necessary to change the purification method by using adifferent affinity purification-tag. In parallel, the ‘‘Additive Screen’’verifies the effect of Ni2+ and Co2+ ions on the protein. This mayguide the choice of a Nickel or Cobalt resin (Table 2, conditionsD9 and D12).

zIt is common to use stabilizing and destabilizing reagentsduring purification that help to reduce aggregation. Metal chela-tors such as ethylenediaminetetraacetic acid (EDTA) at a concen-tration of 1–5 mM avoid metal-induced oxidation of –SH groupsand help to maintain the protein in a reduced state. Moreover,reducing reagents such as beta-mercaptoethanol, dithiothreitol(DTT) and tris(2-carboxyethyl)phosphine (TCEP) can be added

Fig. 8. Different melting curve profiles. (A) Melting curve of esterase shows a sharp and fast thermal denaturation transition within a small temperature interval (55–63 �C).(B) Melting curve of DNA transposase shows good thermal stability, but with a weak denaturation transition (38–65 �C). (C) Example of a multiphase curve suggesting thepresence of a multi-domain protein or a mixture of different proteins. Two overlapping melting curves are observed and the Tm cannot be calculated without ambiguity. (D)Protein precipitation or aggregation can result in a low melting curve intensity. This is also observed for small proteins lacking a hydrophobic core. The resulting curve isnearly flat-shaped and the fluorescence signal is too weak to rely on. (E) The presence of interfering hydrophobic compounds such as detergents, protein aggregates orsolvent-exposed hydrophobic patches can cause high fluorescence background at lower temperatures.


in order to prevent non-specific aggregation mediated by thescrambling of free cysteines forming disulfide bonds. This alsoapplies to proteins that are in vivo in the cytosol, because thereducing environment is not present in vitro. There are proteinsystems that have labile disulfide bonds, and the addition of aparticular reducing agent can shift the equilibrium towards amore stable conformation. For many proteins, it is thereforeimportant to add a small amount of reducing agent to prolongthe life time and monodispersity of the protein. The ‘‘AdditiveScreen’’ helps to identify the concentration limit for such reagentswhere they will not impair protein folding. We have included lowand high concentrations of TCEP (1 and 20 mM) and DTT at a

concentration of 10 mM for comparison, as well as 5 mM EDTA(Table 2, conditions C5–C8 and C12).

Chaotropic agents that can unfold proteins at high concentra-tions can have a stabilizing effect at low concentrations [36].Chaotropic species can rearrange the protein into a more stableconformation, interfering with the intermolecular interactions thatlead to aggregation. Low concentrations of denaturing agents suchas guanidine hydrochloride (GdnHCl) or urea can help to stabilizeproteins with a tendency to aggregate, without affecting thesecondary structure of the protein [37,38]. These chaotropicreagents can be added to purification buffers also to help to reducenon-specific binding. Urea and GdnHCl have been used to study

Table 3Optimization grid for thermal stability assay using SYPRO Orange. Thermofluorexperimental conditions might need to be optimized for some proteins. At the SPCfacility, we used a grid varying the protein concentration versus SYPRO Orangeconcentration. The grid can help to find optimal conditions for the thermaldenaturation assay: which protein/dye ratio gives higher signal to noise ratio,sharper unfolding transition and lower background signal. The conditions used asstandard conditions at the SPC facility are colored in yellow.

0 0.01 ≈ 0.05 0.1 0.25

1X

2.5X

5X

10X

20X

Protein concentration [mg/ml]*

SYPR

O O

rang

e co

ncen

tratio

n*

*Final concentration in 25 µl

aFinal concentration in 25 ll.


global change in protein conformation by differential scanningcalorimetry [39] and to investigate the effect of ligand binding onthe thermal stability in presence or absence of GdnHCl denatur-ation [40]. However, the tolerance for denaturing agents variesfrom protein to protein. In this ‘‘Additive Screen’’, we have includedurea from low to high concentrations (0.1 up to 6 M) where mostproteins become unfolded at room temperature. For comparison,we also included two concentrations of GdnHCl (Table 2, condi-tions A1–A6 and A7–A8).

Crowding agents that mimic the macromolecular crowdingoccurring in a cell have been shown to have a stabilizing effect,and they interfere with the hydration shell of proteins [41]. Glyc-erol is a small polar molecule that can insert itself in the firsthydration shell of proteins. It is also found in many cavities, stabi-lizing the dynamic motions of the side chains surrounding thecavities. Addition of 5–20% glycerol to cell lysate and during puri-fication often contributes to an improved solubility of proteins.Moreover, glycerol is commonly used for protein storage as itprevents unfolding during the freezing step and can be used ascryoprotectant for flash frozen protein crystals. Because glycerolis used for a wide range of applications, we analyze the effect ofglycerol on the protein stability and identify the optimal concen-tration using the ‘‘Additive Screen’’ (Table 2, conditions F2–F5).

Several other crowding agents are included in ‘‘Additive Screen’’in order to cover a wide variety of conditions such as polyethyleneglycols, polyamines, sugars and polyhydric alcohols such as sorbi-tol [33] (Table 2, conditions C9–C11, F6–F9 and G7–G10, respec-tively). Individual nucleotides are abundant in the cell, and theymay bind in any pocket that allows the stacking of an aromaticring. Thus we encourage to test nucleotide binding even if this isnot expected to occur for the protein under investigation. We in-cluded several nucleotides in the ‘‘Additive Screen’’ in the presenceof MgCl2 (Table 2, conditions G11–H12). It is necessary to usefreshly prepared material in order to prevent the degradation ofsensitive nucleotides such as ATP and ADP, and non-hydrolysableanalogues should be considered.

Low concentrations of detergents such as CHAPS or octyl-b-D-glucoside may be useful to prevent protein aggregation and haveshown to be compatible with thermal stability assays using SYPROOrange (Table 2, conditions E11–E12). These detergents have rela-tively small aliphatic tails, and they are sometimes used to solubi-lize membrane proteins. There are many small lipids present in thecytosol of the cell, and these compounds can mimic lipid binding.In addition, we have added spermidine–HCl, which has been asilver bullet for the crystallization of proteins and proteincomplexes [28,42,43] (Table 2, conditions C10–C11). It is worth

to investigate other lipids and detergents if a thermal shift is ob-served for these compounds.

Proteins with a potentially unstable fold tend to be temperaturesensitive, and the unfolding can be slowed down by the addition ofcharged amino acids such as L-arginine and L-glutamic acid. Proteinrefolding studies have shown that the presence of L-arginine in thebuffer inhibits the aggregation of the protein and increases theyield of renatured and biologically active protein [37]. Theamounts used in refolding are substantial (up to 500 mM), and thisis not ideal for structural studies, but it can help during proteinpurification and storage. We include conditions with either L-argi-nine, L-glutamic acid, L-lysine, L-glutamine alone, or L-arginine andL-glutamic acid mixed with concentrations from 50 up to 500 mMin our ‘‘Additive Screen’’ (Table 2, conditions G1–G6).

The ‘‘Additive Screen’’ focuses on small molecules that can affectthe overall stability of the protein during all stages of the proteinproduction process. It can help to review the purification process,and it provides leads to some of the most common protein–ligandinteractions occurring in a cell. Once a trend is established, it isworthwhile to further investigate the effect of a compound withcustomized Thermofluor screens.

Data analysis and melting curve profile

Plotting the melting curve

The analysis of Thermofluor data is based on a plot of the melt-ing curve that represents relative values of the detected fluores-cence intensity coming from the dye versus temperature(Fig. 1A). In case that a constant decrease of the fluorescence is ob-tained before the appearance of the melting peak, we advise to lookat the increase in slope (d(RFU)/dT) (Fig. 1B) and not to look at justthe raw signal. To identify a ligand or a buffer condition that stabi-lizes a protein, the Tm value of the protein under each condition ofthe screen needs to be compared with the reference Tm. When test-ing a large number of conditions in parallel as in the case of the‘‘Buffer Screen’’ and ‘‘Additive Screen’’, we strongly recommend toorganize the data by categories such as pH, salt concentration,divalent ions, nucleotides, etc. Several software packages are avail-able to assist in the data processing as well as in the comprehensi-ble display of the results; we recommend the commerciallyavailable packages GraphPad (http://www.graphpad.com) and Ori-gin (http://www.originlab.com). Alternatively, Frank Niesen fromthe Oxford Structural Genomics Consortium developed custom-made calculation software based on Microsoft Excel to read inThermofluor scans and to compute Tm values from a variety ofavailable RT-PCR instruments [ftp://ftp.sgc.ox.ac.uk/pub/biophys-ics/]. Data processing can be assisted also by software such as Ther-moQ [44]: http://jshare.johnshopkins.edu/aherna19/thermoq/downloads.html.

The reproducibility in thermal shift determination between dif-ferent RT-PCR machines for a particular condition is around <0.2 �Cwhich is much smaller than the thermal shift that can be observedfor the binding of a ligand [4]. When optimizing a buffer, we con-sider shifts in Tm larger than 2 �C significant within a screen wherean experiment has been done under identical conditions. In gen-eral, Tm values are highly reproducible and have shown to varyby less than 2 �C between repeated experiments using separatelypurified samples [28].

Single curve

Since protein unfolding is a cooperative process, the unfoldingof a small protein region will induce the immediate unfolding ofthe remaining protein core; thus an optimal protein stabilization


buffer should result in a sharp and fast thermal denaturation tran-sition between the folded and unfolded state, detected throughhigh transition slopes, in parallel with a higher Tm (Fig. 8A). How-ever, less stable proteins can show a gradual melting curve wherethe protein unfold over a longer temperature range (Fig. 8B). A sin-gle coherently unfolding protein will produce a sigmoidal meltingcurve. The midpoint or inflection point of the transition curve iscalculated using simple equations such as the Boltzmann equation.The melting curve is often followed by a decrease in fluoresenceintensity at higher temperatures. Our interpretation is that a frac-tion of the SYPRO Orange is released gradually from the unfoldedprotein to the aqueous solution where the signal is quenched.However, a residual fluorescence signal remains which might cor-respond to a fraction of SYPRO Orange bound to the denaturatedprotein or to protein aggregates that form at higher temperatures.

Complex curves

Thermal shift assays unfortunately do not always generate sin-gle sigmoidal curves. The presence of a multi-domain protein orthe presence of multiple proteins [25] can lead to a multiphasiccurve suggesting multiple melting transitions (Fig. 8C). Since sepa-rate melting events often overlap, it may be difficult to calculate asingle Tm value. However, many multidomain proteins have do-mains that are sufficiently coupled energetically that they meltin a cooperative manner resulting in a single melting transitionstate [11]. In some cases, we have to assume a two-state unfoldingmodel, and more sophisticated algorithms are needed to fit thesecomplex curves.

Troubleshooting

Although the Thermofluor assay is used as a high throughputmethod, it may require assay optimization for some proteins. Ina systematic case study, approximately 25% of 60 soluble proteinsexamined by Thermofluor using SYPRO Orange were not amenableto such screening, partly due to a high fluorescence background[4]. Recently, the crystallization facility at EMBL-Grenoble per-formed a large scale study on proteins submitted for crystallizationtrials [45]. They measured the thermal stability of 657 soluble pro-teins that entered their facility using SYPRO Orange. Results wereclassified in three categories. In the majority of cases (66%) thesamples produced typical denaturation curves with a clear andsharp temperature transition, allowing a straightforward estima-tion of the Tm. For 30% of the samples, no clear temperature tran-sition was observed, precluding the calculation of a value for theTm. In a few cases complex temperature transitions were observed.Only small differences in crystallization rates were observedbetween the first and the second group indicating that failure toobtain any a denaturation curve should not be interpreted by itselfas a sign that the protein is not feasible for crystallization [45]. Pro-teins with intrinsically disordered regions with a complicated fold-ing landscape are not suitable for Thermofluor, because they givecomplex curves that are often irreproducible. Even if certain trendscan be observed, it is near impossible to interpret them since thestate of the protein over the temperature range and its interactionswith the fluorescent dye are unknown.

Featureless curve

A featureless curve can occur because of problems in the samplepreparation (Fig. 8D), the protocol or the set up of the RT-PCRinstrument. First, we recommend to visually inspect the microplateto see if the protein completely precipitated under the conditionstested. Second, it is advisable to repeat the assay with a higher

protein concentration (40–100 lM): the higher the amount of pro-tein, the cleaner the signal will be as long as the proteins are notunfolded. Increasing the volume of the reaction up to 50 ll willproduce a larger signal, but the strength of the signal is not propor-tional to the increase in volume [28]. If the assay fails again to gen-erate a melting curve, the protein might not contain a hydrophobiccore: we have observed that the proteins that showed no transitionwere often small proteins. In that case, it may be necessary to testthe thermostability of the protein sample by other methods suchas circular dichroism [46] or dynamic light scattering [7]. We alsoobserve in rare cases that the Tm of the protein is higher than themaximum temperature reached on the instrument, so someproteins with an intrinsically high Tm may not be amenable to thisassay. This can be verified by circular dichroism, because that willclearly show if there is a loss in secondary protein structure withinthe experimental temperature range. It can be useful to include apositive control in thermal denaturation assays such as glucoseisomerase or citrate synthase to ensure the experimental set upis correct and the dye is responsive and not degraded. Especiallyif the RT-PCR machine is used for other purposes as well, it isimportant to test whether the machine is calibrated properly forThermofluor before the start of each individual experiment.

High initial fluorescence signal

The highly hydrophobic nature of proteins substantially in-creases the background fluorescence of the assay (Fig. 8E), and inmany cases practically masks the melting transitions [47]. Proteinsdisplaying aberrantly high initial fluorescence in the presence ofSYPRO Orange can be partially unfolded or contain hydrophobicpatches exposed in the native state which might interact withthe dye. It is important to make sure that all aggregated proteinis removed by filtration or centrifugation. It is also possible thatother ingredients in the protein sample (for instance detergents)interact with the dye.

Optimizing the assay

Even if the thermal stability protocol presented above is suit-able for a large number of proteins, some fine tuning might be nec-essary for specific proteins in order to optimize the signal/noiseratio and increase accuracy in the measurement of the Tm. Someparameters can be optimized such as the concentration of SYPROOrange. Kean and coworkers have tested the effect of a range of SY-PRO Orange concentrations and found that the amplitude of signalincreased in a linear fashion with dye concentration. However,they also observed that SYPRO Orange has a destabilizing effectat high concentrations (>20�) [28]. We suggest optimizing 3parameters; the incubation time for the protein/dye mixture priorto the assay, the protein concentration and the dye concentration(Table 3).

Thermofluor applications

Screening the effect of mutations on stability

Protein-engineering techniques such as site-directed mutagen-esis, random mutagenesis and directed-evolution techniques havebeen successfully employed for various proteins. Single amino-acidsubstitutions can have significant impact on the folding and aggre-gation properties of proteins that are both deleterious and favor-able [48–50]. Therefore, the thermal shift assay can be used toquickly pinpoint the effects of specific point mutations on mutantstability relative to wildtype [23,51] or on binding interactions[52]. We present a comparative study (Boivin, unpublished,

Fig. 9. Characterization of the effect of point mutations on the protein stability. Each histogram indicates the thermal shift of a particular point mutation from the meltingtemperature of the wild-type (53.8 �C). Mutations can be either stabilizing or destabilizing compared to the thermal stability of wild-type protein (depending on the aminoacid residue that has been mutated).


Fig. 9) which compares the thermal stability of single pointmutants of a viral protein. The mutants are tested under differentbuffer conditions over a wide pH range to ensure that the thermalshift for the mutant is not simply a result of protein-buffer interac-tions. Indeed, some of the thermal shifts are buffer or pH depen-dent. This figure also illustrates a different way to represent thedata, which is normalized against the wild-type Tm. Thermofluoris ideal to compare the stability of several mutants at the sametime, under identical experimental conditions.

Measuring kinetics of ligand binding by Thermofluor

Thermofluor measures the binding affinity by detecting ligand-dependent changes in the thermal stability of a target protein. Toconfirm that a compound binds and stabilizes the protein in aconcentration-dependent manner, it is possible to perform an as-say in the presence of different concentrations of the compound.While the thermal denaturation assay is used primarily to optimizebuffers and identify ligands, it has been shown that the stabilizingeffect of compounds upon binding is in several cases proportionalto the concentration and affinity of the ligands [4,12,53]. Thermo-fluor has been used successfully to calculate ligand associationconstants and they are in good agreement with isothermal titrationcalorimetry (ITC) measurements [15] as well as radioactive compe-tition and fluorescence polarization assays [11]. For example, Ved-adi and coworkers correlated inhibition data for a protein kinaseand showed that Tm shifts larger than 4 �C translate into valuesfor IC50 < 1 lM. For several compounds, this correlation suggeststhat thermodynamic models adequately describe binding kineticsdespite the irreversible nature of the thermal protein melting[54,55]. This kind of information can for instance be used for pro-tein crystallization, to determine the concentration of a compoundneeded to reach maximum occupancy. Nevertheless, interpreta-tions regarding thermodynamics need to be done with cautionand confirmed by other methods such as isothermal titration calo-rimetry since these affinities depend both on enthalpy and entro-py, which is not properly assessed with Thermofluor [56].

Thermofluor and the crystallizability of protein samples

The homogeneity, stability and solubility of biologicalmacromolecules are key factors that have a strong effect on thepropensity to crystallize. Moreover, crystallization of macromole-cules is a complex procedure which is strongly influenced by di-verse environmental factors such as pH, ionic strength, additives,precipitants, protein concentration and temperature. Identifying

conditions that optimally stabilize proteins in solution has theeffect of reducing the structural heterogeneity of a protein and thispromotes the formation of crystals that are amenable for X-raystructure determination, since crystallization generally favors3-dimensional lattice assembly from structurally identical objects.Ericsson et al., observed a 2-fold increase in the number of crystal-lization hits when proteins were co-crystallized with stabilizingadditives identified with Thermofluor [35]. This suggests thatThermofluor constitutes a valuable and efficient high-throughputmethod to predict and improve the crystallizability of proteins[4,57]. A correlation between complex multiphasic denaturationbehavior and a low likelihood of success in protein crystallizationhas been recently established [45]. Dupeux and coworkers showedin a systematic study that proteins with a Tm higher than 45 �Chave a substantially higher success rate in crystallization thanproteins with lower melting temperatures and proposed to usethe results of this assay to help make rational decisions on whichconstructs or samples to prioritize. Their data also indicated thatcrystallization success rate is maximal when the incubation tem-perature for the crystallization experiments is at least 25 degreesbelow the Tm of the sample determined in the reference buffer con-ditions and advised to set up crystallization trails at 4 �C instead ofroom temperature when the Tm is lower than 45 �C [45].

Concluding remarks

The use of thermal shift assays to monitor protein stability withdifferential fluorescent calorimetry or Thermofluor has become amainstream technique. Originally designed to identify protein li-gands, it has now been developed to improve protein crystallizabil-ity [45], to screen for protein–protein interactions [52] anddifferential modes of protein inhibition [58]. Here we describe theuse of Thermofluor as a general tool to identify stabilizingconditions for recombinant proteins, to improve purification yields,protein storage as well as crystallizability. The two screens pre-sented here test the effect of commonly used chemicals in proteinpurification and storage. The evaluation of these screens providesa quick survey of the overall behavior of the protein, which can beused to improve protein purification and characterization protocols.

Thermofluor online resources

http://www.embl-hamburg.de/facilities/spc.http://Thermofluor.org.https://jshare.johnshopkins.edu/aherna19/thermoq/download.


Acknowledgments

We thank the users of the facility that shared their Thermofluorresults for this manuscript. We would like to thank Sebastian Glattand Christoph Müller (EMBL Heidelberg) for sharing data on Sfc1.We thank the staff of the crystallization platform of EMBL-Heidel-berg and EMBL-Grenoble for constructive discussions. The researchleading to these results received funding from the European Com-munity’s Seventh Framework Program (FP7/2007-2013) undergrant agreement No. 227764 (P-CUBE), and under grant agreementN�283570 (Biostruct-X).

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