This work was supported by Wellcome Trust grant 088419 to MDB.

1. Purification de GTPase de la TPS-étiqueté <ol><li> Culture un E. coli comme BL21 transformées par pGEX-Rac1 O / N à 37 ° C, en agitant à 220 rpm, dans 500 ml de milieu de autoinduction (25 mM Na 2 HPO 4, 25 mM KH 2 PO 4, 50 mM NH 4 Cl, 5 mM de Na 2 SO 4, MgSO 4 2 mM, CaCl2 2 mM, 0,5% de glycerol, 0,05% de glucose, 0,2% de lactose, 5 g de tryptone, 2,5 g d&#39;extrait de levure, 100 pg / ml d&#39;ampicilline). </li><li> Récolte de bactéries par centrifugation pendant 10 min à 10 000 xg, 4 ° C. </li><li> Remettre en suspension culot bactérien dans 20 ml de réactif d&#39;extraction de protéine, inhibiteur de la protéase 1x et incuber pendant 20 min à température ambiante avec inversion. </li><li> Clarifier le lysat par centrifugation à 40 000 g pendant 30 min. </li><li> Ajouter 2 ml de billes magnétiques de glutathion, lavées avec une solution saline tamponnée au phosphate (PBS: 10 mM de Na 2 HPO 4, 1,8 mM de KH 2 PO 4, NaCl 137 mM, KCl 2,7 mM). </li><li> Incuber pendant 2 h, le mélange par inversion à 4 ° C. </li><li> Laver les billes de protéine chargée par quatre fois avec 10 ml de PBS, en utilisant un trieur de particules magnétiques afin de précipiter les billes à chaque étape. </li><li> Billes dans deux ml de PBS et les remettre en suspension la protéine magasin chargé à -80 ° C dans 100 ul de parties aliquotes jusqu&#39;à utilisation. </li></ol> 2. Expression de protéines de liaison à GTPase <ol><li> La veille de l&#39;expérience, des plasmides transfecter codant la protéine fluorescente verte (GFP) -tagged versions de chaque protéine GTPase-contraignant dans un 75-cm 2 ballon séparé de HEK293T comme suit. Pour la validation des nucléotides chargement, transfect GFP-tagged TrioD1 dans un troisième de 75 cm 2 flacon de HEK293T. <ol><li> Diluer polyethylamine à 1 mg / ml dans 100 ul de NaCl 150 mM stérile. </li><li> Ajouter 27 ul polyethylamine diluée à 223 ul réduits médias sériques. </li><li> Ajouter ADN plasmidique de 12 ug à 250 pi réduits médias sériques. </li><li> Incuber chaque tube pendant 2 minutes à température ambiante. </li&gt; <li> Combinez le polyethylamine et l&#39;ADN se mêle dans un seul tube et vortex pendant 2 min. </li><li> Incuber pendant 15-20 min à température ambiante. </li><li> Remplacez le support de croissance (milieu Eagle modifié Dulbecco, 10% de sérum fœtal bovin, 2 mM de L-glutamine, pas d&#39;antibiotiques) sur 90% de confluence HEK293T avec 5 ml de milieu de croissance frais. </li><li> Ajouter le mélange combiné polyethylamine / ADN à incuber la fiole et O / N à 37 ° C, 5% CO 2. </li></ol></li></ol> 3. Purification des protéines de liaison à GTPase <ol><li> Rincer flacons de cellules transfectées dans du PBS et égoutter flacon pendant 5 minutes, aspirer le liquide libre. </li><li> Racler les cellules dans 500 ul de tampon de lyse (50 mM Tris-HCl (pH 7,8), 1% de Nonidet P-40, 1x inhibiteur de protease) en tube de microcentrifugation. </li><li> Lyse des cellules par mélange par inversion à 4 ° C pendant 30 min. </li><li> Au cours de lyse, laver deux lots de 40 ul perles GFP-Trap trois fois avec le tampon de lyse frais, perles sédimentation à 2700 g pendant 2 min entre les lavages. </li><li> Clarifier les lysats par centrifugation à 21 000 xg pendant 10 min. </li><li> Transfert précisé lysat de chacune des protéines de concurrents pour séparer les perles lavées GFP-trap et permettent protéines GFP-fusion pour lier pendant 2 heures, le mélange par inversion à 4 ° C. Gardez lysat de cellules GFP-TrioD1 sur la glace. </li><li> Laver chargés perles GFP-Trap deux fois dans 50 mM Tris-HCl (pH 7,8), 50 mM de NaCl, 0,7% (p / v) Nonidet P-40 et deux fois dans 50 mM Tris-HCl (pH 7,6), mM MgCl 20 2 , sédimentation perles à 2700 g pendant 2 min entre les lavages. </li><li> Éluer les protéines de fusion GFP-40 ul en ajoutant 0,2 M de glycine (pH 2,5) et aspiration et refoulement pendant 30 sec. Immédiatement perles sédiments à 21 000 g pendant 60 s et le liquide de transfert à un nouveau tube de centrifugeuse contenant 4 pi 1 M Tris-HCl (pH 10,4). Le faire rapidement pour limiter les dommages à la protéine purifiée. </li><li> Analyser 1 pi de chaque protéine purifiée par Western blot et la sonde avec un anticorps anti-GFP pour déterminer le rendement relatif en utilisant un Blott quantitativeing système selon le protocole du fabricant. En variante, la détermination des concentrations de protéines par l&#39;acide (BCA) bicinchoninique dosage mais cela introduit des erreurs si les protéines ne réagissent pas avec le dosage d&#39;une manière identique ou il ya protéines contaminantes. </li><li> Égaliser la concentration molaire de la protéine par l&#39;addition de 50 mM de Tris-HCl (pH 7,6), 20 mM de MgCl2. </li></ol> 4. Nucleotide chargement de GTPase <ol><li> Décongeler une aliquote de billes magnétiques TPS-Rac1, préparés à l&#39;étape 1. </li><li> Prendre 90 pl de billes de GST-Rac1 et laver trois fois avec 20 mM de Tris-HCl (pH 7,6), NaCl 25 mM, DTT 0,1 mM, EDTA 4 mM, en utilisant un trieur de particules magnétiques afin de précipiter les billes à chaque étape. </li><li> Aspirer à partir du tampon de perles et ajouter 100 ul de 20 mM de Tris-HCl (pH 7,6), 25 mM de NaCl, 0,1 mM de DTT, 4 mM d&#39;EDTA. </li><li> Selon que le PIB, GTP ou non nucléotidique chargement est nécessaire pour l&#39;expérience de la concurrence, ajouter 12 ul PIB 100 mM, 12 mM ul gu 10anosine 5 &#39;- [γ-thio] triphosphate (GTPyS) ou pas de nucléotides à 60 pi perles TPS-Rac1. </li><li> Pour les contrôles nucléotidique de chargement, les billes restantes divisé en trois aliquotes de 10 ul et ajouter 2 ul de 100 mM de PIB, 2 ul de 10 mM de GTPyS non nucléotidique ou de chaque tube. </li><li> Incuber les mélanges de billes pendant 30 min à 30 ° C avec agitation. </li><li> Stabiliser nucléotidique liée Rac1 par addition de 1 M de MgCl2: 3 pi du mélange à expérimental (étape 4.4), 0,5 pi à chacun des mélanges de commande (étape 4.5). </li></ol> 5. Concurrence contraignant. <ol><li> Mettre en place 6 microtubes, contenant chacun: 200 ul de 50 mM de Tris-HCl (pH 7,6), 20 mM de MgCl2 10 ul expérimentales perles de Rac1 nucléotidiques chargé de l&#39;étape (4.7) 5 ul de protéine Rac1 liaison A (protéine de liaison constant) </li><li> Pour chaque tube, ajouter 0, 1, 2,5, 5, 10 ou 20 ul de protéine B de liaison à Rac1 (protéine de liaison variable). Ces volumes supposent unnviron concentrations d&#39;achat d&#39;actions égales des protéines de liaison constants et variables et peuvent avoir besoin d&#39;être ajustée. <ol><li> Ajustez les volumes de protéines de liaison A et B, si il ya de grandes différences dans les affinités de liaison des deux protéines et cela devrait être déterminées empiriquement à travers les répétitions expérimentales. Porter au volume total du mélange de liaison à 235 ul par addition de 50 mM de Tris-HCl (pH 7,6), 20 mM de MgCl2. </li></ol></li><li> Mettre en place un tube de centrifugeuse contenant: 200 ul de 50 mM de Tris-HCl (pH 7,6), 20 mM de MgCl2 10 ul expérimentales perles de Rac1 nucléotidiques chargé de l&#39;étape (4.7) 10 pi de protéine Rac1 liaison A (protéine de liaison constant) </li><li> Mettre en place le PIB, GTPyS et pas de tubes de contrôle de nucléotides: 200 ul de 50 mM de Tris-HCl (pH 7,6), 20 mM de MgCl2 10 pi de billes de Rac1 de contrôle chargés dans l&#39;étape 4.5 avec le PIB, GTPyS ou non nucléotidique et stabilisées dans l&#39;étape 4.7. 180 pi HEK293T GFP-TrioD1 lysat préparé comme dans l&#39;étape 3.6 4 ul de MgCl2 1 M </li><li> Incuber le mélange pendant 2 heures, le mélange par inversion à 4 ° C. </li><li> Laver les billes trois fois avec 50 mM de Tris-HCl (pH 7,6), 20 mM de MgCl2. </li><li> Éluer les protéines liées dans 20 pi de tampon d&#39;échantillon réducteurs (50 mM Tris-HCl (pH 7), 5% de SDS, 20% de glycerol, 0,02 mg / ml de bleu de bromophénol, 5% β-mercaptoethanol). </li></ol> 6. Analyse de la concurrence <ol><li> Résoudre 10 ul de la protéine liée (étape 5.6) par électrophorèse sur gel de polyacrylamide dodécyl sodium (SDS-PAGE) et transfert de Western. </li><li> Incuber la membrane à 4 ° CO / N en anticorps anti-GFP dilué 1/1000 dans un tampon bloquant dilué dans du PBS 1x à, 0,1% de Tween-20 pour détecter à la fois des protéines de liaison marquées GTPase. </li><li> Laver trois fois la membrane pendant 10 minutes avec du PBS, 0,1% de Tween-20. </li><li> Incuber la membrane pendant 30 minutes à température ambiante dans 800 DyLight conjugué anti-lapin secanticorps secon-, dilué à 1 / 10.000 en tampon de blocage dilué dans du PBS 1x à, 0,1% de Tween-20. </li><li> Laver trois fois la membrane pendant 10 minutes avec du PBS, 0,1% de Tween-20. </li><li> Analyser la membrane en utilisant un système d&#39;imagerie infrarouge, en utilisant le logiciel pour mesurer l&#39;intensité des bandes selon le protocole du fabricant. </li><li> Tracer l&#39;intensité de chaque bande de protéine par rapport au volume du concurrent variable (Protein B). </li><li> Diviser le volume de concurrent variable au point où les lignes se croisent sur le volume d&#39;concurrent constant (protéine A, 5 pi) pour déterminer le rapport de concurrent à laquelle l&#39;équilibre est atteint. </li><li> Pour la validation de l&#39;état de chargement de nucleotides, des membranes de sonde pour p21-activated kinase 1 (PAK1) (un effecteur) et GFP-TrioD1 (a FEM), comme décrit dans les étapes 6.1-6.6. </li></ol>

<ol>
	<li>Burridge, K., Rho Wennerberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=and+Rac+take+center+stage.">and Rac take center stage.</a> Cell. 116 (2), 167-179 (2004).</li><li>Raftopoulou, M., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+Rho+GTPases+lead+the+way.">Cell migration: Rho GTPases lead the way.</a> Dev Biol. 265 (1), 23-32 (2004).</li><li>Rossman, K. L., Der, C. J., Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEF+means+go:+turning+on+RHO+GTPases+with+guanine+nucleotide-exchange+factors.">GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.</a> Nat Rev Mol Cell Biol. 6 (2), 167-180 (2005).</li><li>Worthylake, D. K., Rossman, K. L., Crystal Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+Rac1+in+complex+with+the+guanine+nucleotide+exchange+region+of+Tiam1.">structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.</a> Nature. 408 (6813), 682-688 (2000).</li><li>Scheffzek, K., Ahmadian, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPase+activating+proteins:+structural+and+functional+insights+18+years+after+discovery.">GTPase activating proteins: structural and functional insights 18 years after discovery.</a> Cell Mol Life Sci. 62 (24), 3014-3038 (2005).</li><li>Del Pozo, ., A, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+regulate+GTP-Rac+localized+effector+interactions+through+dissociation+of+Rho-GDI.">Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI.</a> Nat Cell Biol. 4 (3), 232-239 (2002).</li><li>Williamson, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin-1C+and+RCC2+guide+mesenchymal+migration+by+trafficking+Rac1+and+controlling+GEF+exposure.">Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure.</a> J Cell Sci. 127 (Pt 19), 4292-4307 (2014).</li><li>Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+to+the+extracellular+matrix+regulates+the+coupling+of+the+small+GTPase+Rac+to+its+effector+PAK.">Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK.</a> Embo J. 19 (9), 2008-2014 (2000).</li><li>Van Rijssel, J., Hoogenboezem, M., Wester, L., Hordijk, P. L., Van Buul, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-terminal+DH-PH+domain+of+Trio+induces+cell+spreading+and+migration+by+regulating+lamellipodia+dynamics+in+a+Rac1-dependent+fashion.">The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion.</a> PLoS. 7 (1), e29912 (2012).</li><li>Self, A. J., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intrinsic+nucleotide+exchange+and+GTP+hydrolysis+rates.">Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates.</a> Methods Enzymol. 256, 67-76 (1995).</li></ol>

Les auteurs ont rien à révéler.

This protocol describes a method for comparing the relative affinities of pairs of small GTPase-binding proteins. The key steps are the preparation of purified GTPase-binding proteins and the nucleotide loading of the GTPase. The use of GTPase-binding proteins with the same GFP tag, allows the concentrations at which similar amounts of each competitor binds to be accurately determined. The use of recombinant nucleotide-loaded GTPase allows interrogation of the binding properties of the GTPase under specific activity conditions. This step is also the most sensitive as nucleotides will both hydrolyze and detach from the GTPase if the magnesium conditions are not maintained precisely.
In the cell, the large number of GTPase-binding proteins combined with the rapid nucleotide turnover makes such pathways difficult to interpret. The simplicity of this method in comparing only pairs of binding proteins and using carefully controlled nucleotide-loading conditions allows signaling pathways to be elucidated. However, the greatest strength of the protocol is also the greatest weakness as it is a simplification of the in vivo situation. Competition assays can be used to build a robust hypothesis, but this should then be tested in cells by knockdown experiments.
There are three features that must be considered when selecting the GFP-tagged GTPase-binding proteins to be used in the experiment. First, the fusion proteins must express well in mammalian cells, such as HEK293T, as competition assays require a reasonable amount of protein. Second, it must be possible to purify the recombinant protein without significant degradation, and where this is not possible, cloning of a GTPase-binding fragment should be considered. Third, the two GTPase-binding proteins must resolve from one another on SDS-PAGE to allow analysis in section 6.
There are a number of potential caveats to the experiment that need to be considered, and possibly addressed:
Possible denaturation of purified GTPase-binding proteins during the acid elution step or steric hindrance by the GFP tag. In our hands, these have not been a problem, but must be tested. The purified proteins can be tested in functional assays 10. Commercial kits now exist for testing the activity of GEFs or GAPs without the need for isotope-labeled nucleotides. Sequestering proteins, by their nature protect GTPases from GEF or GAP activity, so can be used as competitive inhibitors in the commercial GEF or GAP assays, as we did in our recent publication 7. The relevant feature of proteins that traffic GTPase are the capacity to bind the GTPase, and this can be tested easily in a pull down assay. An alternative approach to testing protein integrity that is applicable to all binding proteins is to titrate protein eluted from GFP-trap beads with glycine with the same protein removed from GFP-trap beads by enzymatic cleavage. The experiment would be analyzed by probing both the GFP-tagged and cleaved protein with an antibody against the protein itself. If the protein is undamaged by elution, equilibrium should be achieved at a 1:1 ratio. This approach would also indicate whether the presence of the GFP tag itself compromises the binding properties of the candidate protein, though this does require the production of a construct with an enzymatic cleavage site between the tag and the binding protein. Whether the protein is compromised by the tag or the elution step, the problem could be addressed by modifying the protocol to use an alternative purification method. Rather than GFP, binding proteins could be His-tagged, purified using Ni-NTA and analyzed using an antibody against the His-tag. The important feature is that both binding proteins must share a common tag although, if necessary, two tags could be added to a protein, one for purification and the other for detection.
The protocol is designed to investigate competition between interactions with the switch I/II domains. Although the majority of GTPase interactions are mediated by this motif, there are some exceptions, most notably the interactions of GDIs that bind to the prenyl tail, as well as obscuring the switch domains. In principle, the protocol could be adapted to use GTPase purified from mammalian cells, so that the GTPase is prenylated, however, the presence of multiple binding sites or allosteric effects complicate the interpretation of competition-binding data. Further problems associated with such a modification are that GDIs co-purify with GTPase from mammalian cells, compromising the purity of the isolated proteins and the hydrophobic nature of the prenyl groups means that prenylated GTPases are associated with either GDI or lipid membrane and such factors would need to be considered in the experiment.
The amount of GST-Rac1 being used in the assay. The constant GTPase binding protein must be at a greater concentration than the Rac1, or when the competitor is added, it will simply bind to free Rac1. It will be immediately obvious if this has happened as binding of the competitor, without a loss of the constant protein, will be detected in much the same way as when the two competing proteins bind to one another as shown in Figure 3B. As an additional control (Step 5.3), a binding reaction containing double the amount of constant binding protein and no variable binding protein should be included (Step 5.3). If the Rac1 in the titration experiment is saturated, doubling the amount of constant binding protein will have no effect on the output. The volumes suggested in the protocol should be appropriate, but the amount of Rac1 can be easily reduced. If binding of the competitor without loss of the constant binding partner is observed, reducing the amount of Rac1 should be attempted before trying to map binding sites to avoid ternary complex formation.
Non-specific interaction of GTPase-binding proteins with the GST or bead, as well as specifically with Rac1. This problem would be manifested by residual binding of the constant GTPase-binding protein, even when the variable GTPase-binding protein has reached a plateau at high concentration. Identification of this issue will be aided by conducting reciprocal experiments where the constant and variable GTPase-binding proteins are swapped. Reciprocal experiments will also greatly improve the accuracy of the estimate of equilibrium point, so should always be included. In cases of non-specific binding, the relative concentrations at which equilibrium is achieved can still be calculated by comparing band intensity between the maxima and minima for each protein, or by measuring the extent of non-specific binding by using GST beads as bait, rather than GST-Rac1.
Pull down assays using different nucleotide-loading conditions should be used to complement the competition assay described in this protocol. Determining the nucleotide preference of partners is important for both understanding the competition events and understanding the signaling pathway that the GTPase-binding protein is involved in. In Figure 2B we analyze binding of proteins with established preference for GTP-loaded or nucleotide-free GTPase as a means to validate nucleotide loading. However, it is sensible to investigate the effect of nucleotide loading on each of the competitors as well. If the hypothetical competitors show different preferences, competition will make less of a contribution to the signaling pathway, and indeed nucleotide turnover is likely to be the mechanism that directs exchange of the binding proteins.

The actin cytoskeleton that determines the shape, polarity and migratory properties of mammalian cells is regulated by the Rho-family of small GTPases. The Rho-family GTPases include RhoA that stimulates cytoskeletal contraction, Rac1 that stimulates actin branching and membrane protrusion, and Cdc42 that has similar effects on actin polymerization to Rac1 and causes the formation of filopodia 1,2. GTPase signaling activity is determined by binding of a nucleotide, which controls the contraction and relaxation of the switch I and switch II loops that mediate the protein-protein interactions with both regulators and effectors. Guanosine 5&#8217;-triphosphate (GTP)-bound GTPases activate downstream effectors, whereas the Guanosine 5&#8217;-diphosphate (GDP)-bound form is inactive. In the cell, cycles of GTP hydrolysis and nucleotide exchange allow rapid turnover of GTPase signals that are necessary for cytoskeletal dynamics. Nucleotide turnover is regulated by three mechanisms. Guanine nucleotide exchange factors (GEFs) stabilize the nucleotide-free GTPase, catalyzing exchange of GDP for GTP, and thereby stimulating GTPase signaling activity 3,4. GTPase-activating proteins (GAPs) catalyze hydrolysis of GTP to GDP, thereby inhibiting GTPase signaling activity 5. Sequestering molecules such as regulator of chromatin condensation 2 (RCC2) and guanine nucleotide dissociation inhibitors (GDIs) obscure the switch loops and in the case of GDIs remove the GTPase from the membrane by interaction with the prenyl tail 6,7. Each of the three classes of regulatory molecule interact with the switch loops, as do the downstream effectors and some trafficking regulators such as coronin-1C 7. The purpose of this protocol is to measure competition for the switch I/II binding site between putative regulators and downstream signaling molecules. It should be noted that competition assays test binding to a shared binding site, so that this protocol is not suitable for testing interactions with other sites, such as binding of GDIs to the prenyl tail.
The subtlety of the conformation differences between active and inactive forms, combined with the labile nature of the bound nucleotide, has made study of GTPase-binding events difficult. The role of the bound nucleotide means that conventional binding assays such as immunoprecipitation or surface plasmon resonance are not well suited to investigation, as the nucleotide cannot be controlled. This obstacle is compounded by the overlap in the binding sites of GEFs, GAPs, effectors, sequestering molecules and trafficking molecules, which make binding data for a single interaction difficult to interpret in the context of the competition that will occur in the cell. Immunoprecipitation, in particular, is compromised by competition between binding partners, as under certain cellular conditions, one binding partner might be identified at the expense of all others, while under other conditions, another partner might dominate. The dynamic nature of GTPase signaling is essential to GTPase function and must be considered when analyzing the relationships between the binding interactions of different regulators. Indeed, we recently described a pathway that relied heavily on competitive binding. We identified coronin-1C as a trafficking molecule that bound to the switch loops of GDP-Rac1 7. In areas of low GEF activity, trafficking would dominate, removing Rac1 from those regions. However, when Rac1 is delivered to regions of the cell where GEF activity is high, the GEF would outcompete coronin-1C, thereby both activating Rac1 and preventing coronin-1C-mediated removal of Rac1 from that area. The model goes further, because the action of the GEF exchanges bound GDP for GTP, shifting the equilibrium still further from coronin-1C. Consequently, Rac1 activity could be explained entirely in terms of competition and relative affinity.
In this protocol, we describe a method for comparing the relative affinities of different binding partners for small GTPases, using Rac1 as an example. By using a purified protein approach, it is possible to piece together a chain of signaling events by pair wise comparison, in an experiment where the bound nucleotide can be closely controlled.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bugbuster</td>
			<td>Novagen</td>
			<td>70584-3</td>
			<td></td>
		</tr>
		<tr>
			<td>COMPLETE protease inhibitor</td>
			<td>Roche</td>
			<td>05 056 489 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione magnetic beads</td>
			<td>Pierce</td>
			<td align="right">88821</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched, average Mw ~25,000</td>
			<td>Sigma Aldrich</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>OPIMEM</td>
			<td>Life Technologies</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10270-1-6</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Trap_A</td>
			<td>Chromotec</td>
			<td>gta-20</td>
			<td></td>
		</tr>
		<tr>
			<td>GDP</td>
			<td>Sigma Aldrich</td>
			<td>G7127</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>GTP&#947;S</td>
			<td>Sigma Aldrich</td>
			<td>G8634</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>Sigma Aldrich</td>
			<td>B6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Living Colors</td>
			<td align="right">632592</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>DyLight 800 conjugated goat anti-rabbit secondary antibody</td>
			<td>Fisher Scientific</td>
			<td align="right">10733944</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAK1 antibody</td>
			<td>Cell Signaling</td>
			<td>2602S</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>Odyssey SA Infrared Imaging System</td>
			<td>Li-cor</td>
			<td>9260-11PC</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparing the affinity of gtpase-binding proteins using competition assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

Ce protocole est conçu pour calculer les affinités relatives des partenaires de liaison pour Rac1, sans avoir besoin de connaître la concentration précise des concurrents (Figure 1). Détermination de la concentration en protéine et introduit des erreurs lors de l&#39;examen compétition entre les molécules dans une voie de signalisation préalable. Cependant, il est important de savoir que les deux concurrents ont la même concentration molaire dans les solutions de réserve pour permettre des rapports simples à calculer lors de l&#39;ajout des volumes différents de l&#39;essai. 40 ul de billes GFP-Trap ont une capacité de liaison de ~ 300 pmol de sorte qu&#39;une confluence de 75 cm 2 flacons de très cellules exprimant se saturer les billes, de sorte que les préparations des deux protéines de liaison différents seront similaires avant l&#39;ajustement (Figure 2A). Si l&#39;on exprime les protéines de mal, ce problème peut être surmonté par la purification de cette protéine à partir de plus d&#39;un flacon de cellules. <p class = &quot;jove_content&quot;&gt; La liaison de la plupart des effecteurs et des régulateurs de GTPase dépend de la nucleotide-chargement de la GTPase de l&#39;appât, il est donc important de tester si le chargement a réussi. Le chargement peut être vérifiée par précipitation de protéines de liaison connus à partir de lysats cellulaires. Protéines effectrices, comme la liaison de GTP PAK1-Rac1 et peuvent être facilement précipité à partir de lysats et détectées par Western blot 8 (figure 2B). GEFS lier préférentiellement au nucléotide sans GTPase de stabiliser l&#39;état de transition. Comme GEF sont de faible abondance, habituellement inactifs et éponger souvent mal, il est préférable de surexprimer une FEM ou d&#39;un fragment du FEM à l&#39;essai gratuitement GTPase nucléotides. Nous utilisons souvent le premier homologie Dbl du Trio, exprimé comme une fusion GFP (GFP-TrioD1 9) (figure 2B), mais toute FEM travailler. Les protéines qui se lient à la GTPase de PIB chargé sont plus rares. Nous avons récemment rapporté RCC2 comme l&#39;une telle protéine 7, ou le PIB de chargement peut être validé simplement comme binding à FEM ni ni effecteur. La sortie à partir de l&#39;expérience sera un transfert de Western illustrant les deux partenaires de liaison marqués GFP liés à la GTPase. En utilisant un seul anticorps pour détecter les deux protéines, les concentrations auxquelles des quantités similaires des deux concurrents se lient peuvent être déterminées et donc les affinités relatives déduites. Dans cet exemple, la concurrence entre le domaine de l&#39;hélice de la protéine Rac1-trafic, coronine-1C (Rac1-binding protein A), et la protéine Rac1-séquestrant, RCC2 (Rac1-binding protein B), est démontré (figure 3A). En utilisant un volume constant de coronine-1C hélice (5 pi), et en ajoutant des volumes croissants de RCC2, nous pouvons voir de la GFP efface que l&#39;équilibre est atteint à 1,25-2,5 ul de RCC2 (astérisque), démontrant que RCC2 a une plus forte affinité pour Rac1 que coronine-1C. En mesurant l&#39;intensité des bandes en utilisant quantitative Western blot, et le tracé des valeurs moyennes pour chaque competitor, le point d&#39;équilibre peut être calculée avec précision, en identifiant les volumes à laquelle se coupent les courbes (figure 3B). L&#39;un des obstacles possibles à un dosage de compétition est réussie si les partenaires de liaison se lient les uns aux autres ainsi que la liaison à Rac1. Dans la figure 3A + B nous démontrons la concurrence entre RCC2 et le domaine de l&#39;hélice de coronine-1C, plutôt que sur toute la longueur coronine-1C. La raison pour utiliser le coronine tronquée est que coronine-1C se lie également RCC2 à travers le domaine de la queue. Lorsque pleine longueur coronine-1C est titré contre RCC2, la liaison de deux protéines est détectée, en raison de la formation complexe ternaire, plutôt que la concurrence (figure 3C). Si la concurrence est en cours, la liaison d&#39;une protéine va augmenter tandis que les autres baisses, et lié total GFP-fusion restera constant. Dans les cas où un complexe ternaire se forme, il est nécessaire de tronquer une de la protéine GTPase se liant de sorte que la competvisi- plus interagir. <img alt="Figure 1" src="/files/ftp_upload/53254/53254fig1.jpg" /> Figure 1. Workflow. Représentation schématique du flux de travail pour déterminer l&#39;affinité des protéines de GTPase liaison en utilisant des analyses de la concurrence. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 2" src="/files/ftp_upload/53254/53254fig2.jpg" /> Figure 2. La validation des protéines purifiées. (A) purifiée GFP-étiqueté protéines analysées par transfert de Western de liaison, sondant avec anti-GFP pour déterminer le rendement relatif des deux protéines Rac1. Ce type d&#39;égalisation pendant l&#39;expérience permet la concentration des deux protéines à être réglé de façon qu&#39;ils correspondent à l&#39;expérience de liaison. (B) GDP, GTPyS et aucun nucléotide chargé TPS-Rac1 a été incubé avec un lysat de HEK293T exprimant la GFP-TrioD1 et protéines détectées en traçant pour PAK1 endogène ou surexprimé GFP-TrioD1 liés. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig2large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 3" src="/files/ftp_upload/53254/53254fig3.jpg" /> Figure 3. Analyse par Western blot de la protéine par rapport Exemple sorties contraignant. À partir des essais de compétition de liaison. (A) PIB chargé Rac1 a été mélangé avec 5 pi GFP-coronine-1C domaine de l&#39;hélice et de l&#39;augmentation des volumes de GFP-RCC2 ont été titrés en jeu. En Western blot des protéines liées à GFP, des problèmes avec détection différentielle des deux protéines sont évités et le signal de la GFP indique le rapport molaire entre les deux protéines de fusion. Les astérisques indiquent les rapports de concurrence sur eithecôté r du point d&#39;équilibre. (B) des intensités de bande des protéines liées à la GFP de fusion de trois expériences indépendantes ont été mesurées par quantitative Western Blot, en utilisant des anticorps et moyennes secondaires fluorophores conjugué comploté pour calculer le montant de RCC2 nécessaire pour atteindre l&#39;équilibre. (C ) Exemple de sortie à partir d&#39;une expérience dans laquelle des protéines de liaison à Rac1 lient les unes aux autres et forment un complexe ternaire, au lieu de la compétition. PIB chargé Rac1 a été mélangé avec 5 pi GFP-RCC2 et l&#39;augmentation des volumes de GFP-coronine-1C pleine longueur ont été titrés. L&#39;augmentation liée GFP-coronine-1C sans perte de borne GFP-RCC2 indique formation d&#39;un complexe ternaire. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig3large.jpg" target="_blank">S&#39;il vous plaît cliquez ici pour voir une version plus grande de cette figure.</a>

Watch this Scientific Journal Video about Comparing the Affinity of GTPase-binding Proteins using Competition Assays at JoVE.com

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

En comparant l&#39;affinité des protéines de liaison en utilisant GTPase Compétition dosages

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

comparing-affinity-gtpase-binding-proteins-using-competition

Research

JoVE Journal

Biology

9.0K vues.  University of Bristol. This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

Vidéo: Comparing the Affinity of GTPase-binding Proteins using Competition Assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analyse de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Recherche

Biologie

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

La cristallisation des protéines membranaires dans des mésophases lipidique

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Haut débit cristallisation des protéines membranaires en utilisant la méthode lipidique Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identification des partenaires protéine interagissant Utilisation Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

À haut débit Affinity Purification de protéines recombinantes-étiquette

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

TPS-Sa purification: A deux pas d&#39;affinité Protocole Purification Cédant longs protéines purifiées

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In Vitro agrégation essais utilisant la protéine tau hyperphosphorylée

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Étudier les protéines prions comme épandage et la toxicité de l&#39;utilisation des Métazoaires organisme modèle<em&gt; C. elegans</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Identification de MyoD Interactome Utilisation Tandem Affinity Purification couplée à la spectrométrie de masse

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...