Esta investigación fue financiada por una beca de Wellcome Trust Superior otorgado al Dr. Ian Goodfellow.

1. Generación de líneas celulares: pMEP4 transfección / expresión <ol><li> Transfectar células con el vector de expresión pMEP4 y seleccionar con 100 g de higromicina B / ml (Roche) hasta que todas las células transfectadas simuladas se han matado. El plásmido pMEP4 se mantiene episomalmente lo que no hay necesidad de aislar clones específicos. </li><li> Las células que contienen el vector pMEP4 puede ser inducida por tratamiento con 10 CdCl 2 mM durante 16 horas. Expresión y la inducibilidad debe ser confirmada en una pequeña escala antes de la amplificación de las líneas celulares. La proteína TAP-etiquetados puede ser fácilmente detectado como los dominios de la proteína G se unen a los anticuerpos de casi todas las especies. Para los grandes purificaciones normalmente 10 confluentes 175 cm 2 frascos de células son obligatorios. Esto equivale a aproximadamente 2 X10 8 células que expresan. </li></ol> 2. De preparación de células lisado <ol><li> Raspe las células en PBS y se combinan en un tubo de 50 ml de una sola. Spin 1200X gfo 5 minutos (esto debería resultar en 2-3 ml de células empaquetadas para empezar, lo que reduce a aproximadamente 1,5 ml después del lavado). </li><li> Se lavan las células tres veces en PBS enfriado en hielo (50 ml cada vez). </li><li> Lisar las células en 5 ml de tampón de lisis (50 mM Tris-HCl (pH 7,5), NaCl 125 mM, glicerol al 5%, 0,2% de NP-40, 1,5 mM de MgCl 2, 25 mM de NaF, 1 mM Na 3 VO 4 y el inhibidor de proteasa ). Nota: NaF, Na 3 VO 4 y los inhibidores de la proteasa debe añadirse fresco. Pipetear arriba y hacia abajo 10 veces antes de dejar durante 5 minutos en hielo. Veces jeringa hacia arriba y hacia abajo 5-10 usando una aguja roma antes de salir de nuevo en hielo durante 5 minutos en hielo. Repetir su inyección y se deja durante otros 5 minutos en hielo. </li><li> Congelación-descongelación del lisado dos veces (hielo líquido N / 2 o seco y etanol). No permita que la muestra para llegar a más de 4 ° C. Nota: se puede almacenar la muestra a -80 ° C hasta que tenga tiempo para procesar. </li><li> Alícuotas de la muestra en tubos de 1,5 ml y eliminar las células no lisadas y debris por centrifugación (10 minutos a 4 ° C, 16.000 xg). </li><li> Recuperar sobrenadante, se combinan en un tubo Falcon de 15 ml y (opcionalmente) pasan a través de unos 0,45 m de filtro antes de tomar una muestra de 20 l para la cuantificación de proteínas rendimiento. Eliminar un ulterior 50 l alícuota para su posterior análisis (Muestra 1). </li></ol> 3. La unión a IgG de conejo-agarosa (Nota: Todos los giros deben llevarse a cabo a 1200X g en una centrífuga refrigerada a 4 ° C durante 1 minuto, a menos que se indique lo contrario) <ol><li> Resuspender la IgG de conejo-agarosa solución (Sigma-Aldrich) por agitación botella. Tomar 380 l de IgG de agarosa (utilizando un corte de punta de la pipeta 1 ml) y retirar el envío / solución conservante por centrifugación durante 1 minuto. Lavar la agarosa tres veces en tampón de lisis frío (4 ° C) usando centrifugación para aclarar. El rendimiento final de agarosa debe ser de aproximadamente 250 l de perlas envasados. </li><li> Añadir el lisado celular desaparecido de 2,6 (en una tina de 15 ml Falcone) el lavado de conejo IgG de agarosa y se incuba durante 3 horas (o durante la noche) a 4 ° C usando un mezclador giratorio. </li><li> Derivado establecen las perlas de agarosa durante 5 minutos a 4 ° C y separar el sobrenadante para su posterior análisis (Muestra 2). Nota: El TAP etiquetado de proteínas debe ahora estar asociados con las cuentas. </li></ol> 4. Escisión de la proteasa TEV (Nota: Todos los giros deben llevarse a cabo a 1200X g en una centrífuga refrigerada a 4 ° C durante 1 minuto, a menos que se indique lo contrario) <ol><li> Lavar las perlas de agarosa IgG de conejo tres veces en tampón de lisis frío (4 ° C) mediante centrifugación para aclarar (nota: el tampón de lisis no debe contener inhibidores de la proteasa). Se debe tener cuidado de no eliminar o perder los granos durante los pasos de lavado. </li><li> Se lavan las perlas de otras dos veces con tampón de escisión VET-proteasa (10 mM Tris-HCl (pH 7,5), NaCl 100 mM, y 0,2% NP-40) utilizando centrifugación para aclarar. Después del último lavado retire con cuidado todas las liquid de las perlas. </li><li> Prepare una mezcla de la proteasa TEV división, para cada muestra se incluyen 467,5 l H 2 O, 25 l 20x TEV buffer (Invitrogen), 5 ml DTT 0,1 M y 2,5 l (25 U) de la proteasa TEV (Invitrogen). Añadir 500 l de esta mezcla de escisión VET a cada muestra de perlas envasados ​​y transferir toda la mezcla a un 1,7 ml de pre-lubricado tubo (Costar). Antes de la incubación eliminar una parte alícuota de 30 l para su posterior análisis (Muestra 3). </li><li> Incubar la reacción proteasa TEV durante la noche a 4 ° C usando un mezclador giratorio. Nótese que tiempos más cortos incubaciones se pueden utilizar dependiendo de la naturaleza de la proteína cebo y la accesibilidad del sitio de escisión de la proteasa VET pero el tiempo de incubación mínimo debe ser determinado empíricamente. </li></ol> 5. La unión a estreptavidina inmovilizada Ultralink Plus cuentas (Nota: Todos los giros deben llevarse a cabo a 1200X g en una centrífuga refrigerada a 4 ° C durante 1 minuto, a menos que se indique lo contrario) </p&gt; <ol><li> Centrifugar la reacción de escisión VET que contiene las perlas de agarosa IgG de conejo durante 5 minutos a 1200X g (4 ° C). Retirar una parte alícuota de 20 l del sobrenadante clarificado para el análisis (Ejemplo 4) </li><li> Transferir el sobrenadante restante (aproximadamente 480 l) a un nuevo máximo de 1,5 ml de pre-lubricadas tubo (Costar) y dejar este en el hielo. No se deshaga de este sobrenadante - contiene la proteína de TEV cebo hendidas y las proteínas de unión asociados. </li><li> Añadir 500 l de tampón de lisis frío (Sección 2.3) a las cuentas restantes de IgG de conejo de agarosa y volver a suspender. Centrifugar esta mezcla para aclarar las perlas antes de retirar el sobrenadante y combinándolo con el de paso 5,2. Deje este tubo en hielo (que ahora debe contener ~ 980 ml de muestra). </li><li> Conserve las IgG de conejo perlas de agarosa para su posterior análisis (muestra 5). Nota: si se ejecuta este ejemplo en un gel de SDS-PAGE (o Western Blot) que le dará un montón de fondo debido a la presencia de la pesada de IgG y de la luzcadenas. </li><li> Mientras tanto Resuspender el Ultralink estreptavidina inmovilizada Plus perlas de agarosa (Pierce), haciendo girar la botella. Con una punta de pipeta de 200 ml con el corte final, alícuota de 70 l de cuentas estreptavidina por muestra en pre-lubricadas tubos de 1,5 ml y eliminar el envío / solución conservante por centrifugación. Nota: estas cuentas son muy pequeñas y por lo tanto &quot;pico de pato&quot; o plana / estrecho de composición consejos pueden ser utilizados para minimizar la pérdida de grano durante el lavado. </li><li> Lávese las cuentas estreptavidina tres veces en tampón de lisis frío (4 ° C) mediante centrifugación para aclarar. Esto debería dejar de aproximadamente 45-50 l de bolas llenas por tubo. </li><li> Transferir el sobrenadante escisión VET-proteasa (es decir, el l de la muestra 980 de paso 5,3) al tubo que contiene las perlas de estreptavidina lavadas e incubar durante 3 horas (o durante la noche) a 4 ° C usando un mezclador giratorio. </li><li> Derivado establecen las perlas de estreptavidina durante 5 minutos a 4 ° C y separar el sobrenadante para su posterior análisis (Samejemplo 6). Lávese las cuentas estreptavidina tres veces en tampón de lisis frío (4 ° C) mediante centrifugación para aclarar. Después del lavado final, cuidadosamente eliminar el tampón de lavado restante. </li></ol> 6. Biotina elución del péptido y proteína de unión de estreptavidina cebo (Nota: Todos los giros deben llevarse a cabo a 1200X g en una centrífuga refrigerada a 4 ° C durante 1 minuto, a menos que se indique lo contrario) <ol><li> Eluir proteínas etiquetadas de las perlas de estreptavidina mediante la adición de 500 l de PBS: biotina (1 mM de D-biotina) y la incubación a 4 ° C durante 3 horas (o durante la noche) usando un mezclador giratorio. </li><li> Gira por las perlas de estreptavidina durante 5 minutos a 4 ° C y retirar el sobrenadante a un tubo de 1,5 ml de pre-lubricadas (Nota: esta es la muestra final / de elución (Ejemplo 7). </li><li> Añadir otra Biotina 500 l: PBS a las perlas de estreptavidina restantes y se incuba a 4 ° C durante 2-3 horas (o durante la noche) usando un mezclador giratorio. </li><li> Returba el paso 6.2 y combinar los dos eluciones (Ejemplo 7). Este elución final puede ser almacenado a -80 ° C. </li><li> Para evaluar la eficiencia de la elución de la biotina, congelar las cuentas de estreptavidina restantes y hervir más tarde en tampón de muestra SDS (Muestra 8) (recomendaciones: LaneMarker 5x tampón de muestra de la reducción de Fisher). </li></ol> 7. La concentración de proteínas <ol><li> El eluido final (Ejemplo 7) tiene que ser concentrado antes de su análisis, debido a la baja concentración de proteínas y el volumen relativamente alto. Esto se puede lograr utilizando de bajo peso molecular (menos de 5 kDa) columnas Vivaspin spin (Vivascience). Por objeto concentrar el volumen a menos de 100 l. Esta muestra final se puede hervir y se almacena en el búfer de la proteína de la muestra (recomendaciones: LaneMarker 5x tampón de muestra de la reducción de Fisher). </li></ol> 8. Análisis <ol><li> Las muestras 1-8 se puede analizar por Western blot para determinar la eficacia de tirar hacia abajo. Nota: La mayoría de los anticuerpos secundarios seuna reacción cruzada con los dominios de la proteína G de la proteína de fusión, pero esto se elimina después de la escisión de la proteasa VET. </li><li> Ejemplo 7 pueden ser analizados por 1D o 2D SDS-PAGE. La tinción puede realizarse usando plata o de Coomassie (recomendaciones: tinción SilverQuest plata coloidal y kits de tinción con azul Coomassie de Invitrogen). Identificación de proteínas se puede realizar utilizando espectrometría de masas. </li></ol> 9. Los resultados representativos Un ejemplo de 1D análisis SDS-PAGE de la elución final (Ejemplo 7) de este protocolo para identificar parejas de unión de eIF4E TAP etiquetado se proporciona en la Figura 2. Este gel representativo refleja la naturaleza compleja y abundante de las interacciones con otras proteínas eIF4E en la célula. La comparación con el control negativo también se muestra en la Figura 2, generada a partir de una línea celular que expresa sólo la etiqueta TAP, ilustra la especificidad de este eIF4E cebo desplegable. En este ejemplo el 15% de la elución final concentrada (Muestra7) se analizó por SDS-PAGE 1D utilizando comercialmente disponibles prefabricados geles de gradiente antes de ser teñidos utilizando el kit de tinción de plata Silverquest de Invitrogen. Típicamente 50-85% de la restante elución concentrado final (Ejemplo 7) se analiza con Coomassie coloidal mancha (Invitrogen). Las muestras (rodajas de gel o bandas) de todo el carril se extrajeron luego y se analizaron por espectrometría de masas. Las proteínas identificadas en el eIF4E pull-down se filtraron en contra de las parejas de unión del control negativo para identificar no específicos asociados vinculantes. Las proteínas finales identificados utilizando este proceso de eIF4E TAP etiquetado puede verse en la Figura 2, que es representativo de las proteínas normales identificados utilizando esta técnica. Además, un número de las subunidades eIF3 También se han detectado (datos no presentados). <img src="/files/ftp_upload/3643/3643fig1.jpg" alt="Figura 1" /> Figura 1. Esquema de laconjuntamente procedimiento de purificación por afinidad. El tándem de seis pasos de purificación de afinidad (TAP) de protocolo implica la generación de líneas celulares, la lisis celular, IgG de conejo de agarosa inmuno-precipitación, TEV división de la proteasa, estreptavidina purificación por afinidad de cuentas y, finalmente elución biotina. <img src="/files/ftp_upload/3643/3643fig2.jpg" alt="Figura 2" /> Figura 2. Tándem purificación por afinidad de la proteína murina eIF4E. Interactuar socios de la N terminal TAP etiquetados eIF4E se purificaron a partir de células eucariotas HEK293 utilizando el protocolo adjunto. Una fracción del 20% de la elución final (Ejemplo 7) se analizó por SDS-PAGE en un gel de gradiente prefabricado 4-12% (PAT-eIF4E carril). Un análisis equivalente se llevó a cabo para la etiqueta por sí solo (carril TAP). Las proteínas se identificaron mediante la tinción de plata. Las proteínas posteriormente identificados por espectrometría de masas de la misma muestra se destacan en este gel. Abreviaturas: eIF4G; iniciación de la traducción eucariótica factor de 4 gamma, PABP, proteína poli vinculante, eIF4A: factor de iniciación de la traducción eucariótica 4 alfa, SBP-eIF4E, la proteína PAT cebo restante que contiene el péptido estreptavidina vinculante fusionado con el factor 4E traducción eucariótico de iniciación, 4EBPs; eucariótico de iniciación de traducción 4E factor de unión proteínas, eIF4eNiF1l eucariótico de iniciación de traducción factor de factor nuclear de importación 4E 1, PAS, el péptido estreptavidina restante de unión del péptido TAP contenga restos de TEV.

<ol>
	<li>Williamson, M. P., Sutcliffe, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions.">Protein-protein interactions.</a> Biochem. Soc. Trans. 38 (4), 875-878 (2010).</li><li>Rigaut, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat. Biotechnol. 17 (10), 1030-1032 (1999).</li><li>Blackwell, C., Brown, 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+application+of+tandem-affinity+purification+to+Candida+albicans.">The application of tandem-affinity purification to Candida albicans.</a> Methods Mol. Biol. 499, 133-148 (2009).</li><li>Cochrane, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+complex+formation+between+HIV+Rev+and+the+nucleosome+assembly+protein,+NAP1,+affects+Rev+function.">Stable complex formation between HIV Rev and the nucleosome assembly protein, NAP1, affects Rev function.</a> Virology. 388 (1), 103-111 (2009).</li><li>Fernandez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+tandem+affinity+purification+of+PSD-95+recovers+core+postsynaptic+complexes+and+schizophrenia+susceptibility+proteins.">Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.</a> Mol. Syst. Biol. 5, 269-269 (2009).</li><li>Gloeckner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+affinity+purification+of+protein+complexes+from+mammalian+cells+by+the+Strep&#47;FLAG+(SF)-TAP+tag.">Tandem affinity purification of protein complexes from mammalian cells by the Strep&#47;FLAG (SF)-TAP tag.</a> Methods Mol. Biol. 564, 359-372 (2009).</li><li>Holowaty, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+profiling+with+Epstein-Barr+nuclear+antigen-1+reveals+an+interaction+with+the+herpesvirus-associated+ubiquitin-specific+protease+HAUSP/USP7.">Protein profiling with Epstein-Barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease HAUSP/USP7.</a> J. Biol. Chem. 278 (32), 29987-29994 (2003).</li><li>Burckstummer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+tandem+affinity+purification+procedure+for+interaction+proteomics+in+mammalian+cells.">An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.</a> Nat. Methods. 3 (12), 1013-1019 (2006).</li><li>Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calicivirus+translation+initiation+requires+an+interaction+between+VPg+and+eIF+4+E.">Calicivirus translation initiation requires an interaction between VPg and eIF 4 E.</a> EMBO Rep. 6 (10), 968-972 (2005).</li><li>Goodfellow, I. G., Roberts, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+initiation+factor+4E.">Eukaryotic initiation factor 4E.</a> Int. J. Biochem. Cell Biol. 40 (12), 2675-2680 (2008).</li></ol>

No hay conflictos de interés declarado.

La técnica de marcado TAP ilustrado aquí demuestra un método altamente específico y estrictas para aislar las parejas de unión de proteínas de cebo en las células eucariotas. Este enfoque puede ser aplicado a las proteínas celulares y virales. A nuestro entender, esta es la primera vez que esta técnica aplicada a la traducción eIF4E factor de iniciación. Identificación de la eIF4G eIF4E conocido proteínas vinculantes y los 4EBPs uso de esta técnica se confirma la validez de este enfoque. Además, la identificación del componente restante del complejo eIF4F, a saber eIF4A, y PABP confirma que la interacción indirecta y complejos terciarios permanecer intacto durante el proceso de purificación. La identificación de múltiples isoformas de las proteínas de unión eIF4E canónicos era también evidente. Estos se describen con más detalle en la Figura 2. En cuanto a las limitaciones del enfoque, la atención de algunos se debe tomar con respecto a la elecciónde proteína cebo y si o no para colocar la etiqueta TAG en el extremo N-o C-terminal. Podría ser conveniente para llevar a cabo un ensayo funcional o examinar la localización de la proteína de fusión con el microscopio antes de la purificación a gran escala para asegurar el derivado de etiquetado es funcional. Integral de la membrana o proteínas nucleares no necesariamente puede ser liberado por las condiciones relativamente suaves detergentes descritas en la etapa de lisis. Como con todas las inmunoprecipitaciones y similares ensayos desplegable, se podría hacer modificaciones a la naturaleza y concentración del detergente en el tampón de lisis para aumentar la eficiencia de la lisis. La concentración iónica de los tampones de lisis y lavado también puede modificarse para aumentar o disminuir la severidad de la purificación. También es posible llevar a cabo la purificación inicial bajo condiciones suaves estándar (descrito anteriormente), pero posteriormente dividir las perlas de estreptavidina (sección 5,8) en alícuotas de 4-5, que posteriormente se pueden lavar bajo con iónica crecientecondiciones. Esto puede permitir al usuario identificar las condiciones óptimas que eliminan proteínas no específicas que interactúan. Todo el proceso puede realizarse también utilizando la transfección transitoria cuando las distintas partes que interactúan son conocidos y su presencia o ausencia determinó por transferencia subsiguiente occidental solamente. En este caso, le sugerimos dos 100cm 2 placas de células transfectadas con cada uno de 8 g de plásmido de expresión. Este procedimiento modificado es de uso particular, al examinar la capacidad de los derivados mutantes de la proteína TAP fusión para interactuar con parejas de unión conocidas. El análisis de las muestras 1 a 8 en la teñidos con plata geles SDS-PAGE (o por Western blot, si un anticuerpo está disponible) es una excelente forma de solución de problemas, si la técnica de no generar resultados aceptables. La eficacia de cada precipitación basado en la afinidad y la elución específica puede ser analizado utilizando este método de muestreo sistemático. Las muestras de uno a ocho se debe tomar durante Optimzación del protocolo para cada nuevo cebo. La técnica de marcado TAP ofrece una alternativa potente y robusta con otros enfoques, tales como GST pull-downs, la levadura de dos ensayos de híbridos, etc La purificación por afinidad de matrimonio y los pasos específicos de elución (TEV división y la elución biotina) proporcionan la especificidad y el rigor que se mantiene a un alto nivel durante todo el procedimiento de purificación. Críticamente esta técnica se puede aplicar a cualquier proteína y por lo tanto representa un método excelente para la identificación de parejas de unión para una proteína diana de interés.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Higromicina B </td><td> Roche </td><td> 10843555001 </td></tr><tr><td> IgG de conejo de agarosa </td><td> Sigma </td><td> A2909 </td></tr><tr><td> AC-VET proteasa </td><td> Invitrogen </td><td> 12575-015 </td></tr><tr><td> Cóctel de inhibidores de la proteasa </td><td> Calbiochem </td><td> 539134 </td></tr><tr><td> Ultralink estreptavidina inmovilizada cuentas Plus </td><td> Atravesar </td><td> 53116 </td></tr><tr><td> Vivaspin 500 concentrador centrífugo (5KDa) </td><td> Vivaspin </td><td> V50112 </td></tr><tr><td> SilverQuest kit de tinción de plata </td><td> Invitrogen </td><td> LC6070 </td></tr><tr><td> Novex azul de Coomassie coloidal kit de tinción </td><td> Invitrogen </td><td> LC6025 </td></tr><tr><td> 1,7 ml tubos prelubricados</td><td> Costar </td><td> 3207 </td></tr><tr><td> Microcapilar puntas de pipeta </td><td> VWR </td><td> 37001-150 </td></tr><tr><td> NuPage 4-12% Bis-Tris gradiente de geles </td><td> Invitrogen </td><td> NP0322BOX </td></tr><tr><td> CdCl 2 </td><td> Sigma </td><td> 202908 </td></tr><tr><td> 5x tampón de muestras SDS </td><td> Pescador </td><td> PN39000 </td></tr></tbody></table>

identification of protein interacting partners using tandem affinity purification

Un paso crítico y la limitación de frecuencia en la comprensión de la función de acogida y las proteínas virales es la identificación de la interacción de proteínas asociados celulares o virales. Existen muchos enfoques que permiten la identificación de socios que interactúan, incluyendo la levadura de dos sistema híbrido, así como derribar ensayos utilizando proteínas recombinantes y de inmunoprecipitación de las proteínas endógenas seguido por espectrometría de masa de identificación 1. Estudios recientes han puesto de relieve la utilidad de la doble purificación por afinidad mediada por la etiqueta, junto con dos pasos de elución específicos en la identificación de proteínas que interactúan. Este enfoque, denominado tándem Purificación por afinidad (TAP), se utilizó inicialmente en la levadura 2,3 pero más recientemente se ha adaptado para usar en células de mamífero 4-8. Como prueba de concepto que hemos establecido una purificación por afinidad en tándem (TAP), utilizando la bien caracterizada iniciación de la traducción eucariótica factortor eIF4E 9,10. La traducción eIF4E factor celular es un componente crítico del complejo eIF4F celular implicada en la tapa que dependen de iniciación de traducción 10. La etiqueta TAP utilizado en el presente estudio se compone de dos unidades de proteína G y un péptido de unión de estreptavidina separados por un virus del Tabaco Etch (VET) secuencia de escisión de la proteasa. La etiqueta TAP utilizado en el presente estudio se compone de dos unidades de proteína G y un péptido de unión de estreptavidina separados por un virus del Tabaco Etch (VET) secuencia proteasa escisión 8. Para renunciar a la necesidad de que la generación de líneas celulares clonales, hemos desarrollado un sistema rápido que se basa en la expresión de la proteína TAP-etiquetados cebo de un plásmido episomal mantenido sobre la base de pMEP4 (Invitrogen). Expresión de eIF4E etiquetados murino de este plásmido se controla mediante el cloruro de cadmio promotor inducible metalotioneína. La lisis de las células que expresan y purificación de afinidad posterior a través de la unión a Rabbit IgG de agarosa, la escisión VET proteasa, la unión a estreptavidina ligada agarosa y elución biotina posterior identificado numerosas proteínas aparentemente específicas para el eIF4E desplegable (en comparación con el control de líneas celulares que expresan la etiqueta TAP solo). Las identidades de las proteínas se obtuvieron mediante la escisión de las bandas de espectrometría 1D tándem SDS-PAGE y posterior masa. Los componentes identificados incluyen las proteínas de unión eIF4E conocidos eIF4G y 4EBP 1. Además, otros componentes del complejo eIF4F, de los cuales eIF4E es un componente fueron identificados, a saber eIF4A y Poly-Una proteína de unión. La capacidad de identificar no sólo se conocen los socios directos de unión, así como secundarias proteínas que interactúan, destaca aún más la utilidad de este enfoque en la caracterización de proteínas de función desconocida.

Watch this Scientific Journal Video about Identification of Protein Interacting Partners Using Tandem Affinity Purification at JoVE.com

Identification of Protein Interacting Partners Using Tandem Affinity Purification

Tandem purificación de afinidad es un enfoque sólido para la identificación de socios de la unión a proteínas. Como prueba de concepto, esta metodología se aplicó a la traducción factor eIF4E bien caracterizado iniciación a co-precipitado los factores implicados en la célula huésped iniciación de la traducción. Este método se adapta fácilmente a cualquier proteína celular o viral.

Identificación de los socios interacción de proteínas en tándem de purificación de afinidad

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

identification-protein-interacting-partners-using-tandem-affinity

Research

JoVE Journal

Biology

37.4K vistas.  Imperial College London. Tandem purificación de afinidad es un enfoque sólido para la identificación de socios de la unión a proteínas. Como prueba de concepto, esta metodología se aplicó a la traducción factor eIF4E bien caracterizado iniciación a co-precipitado los factores implicados en la célula huésped iniciación de la traducción. Este método se adapta fácilmente a cualquier proteína celular o viral.

Video: Identification of Protein Interacting Partners Using Tandem Affinity Purification

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Investigación

Biología

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

La purificación de Hsp104, una proteína Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificación de proteínas ortogonales Facilitado por un marcador de afinidad biespecíficos pequeños

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Identificación y caracterización de la glicosilación de proteínas específicas de uso de endo-y exoglicosidasas

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificación de los complejos de proteínas en<em&gt; Escherichia coli</em&gt; La purificación mediante afinidad del péptido secuencial en combinación con espectrometría de masas tándem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

El Protocolo para la identificación de las interacciones proteína-proteína base de<sup&gt; 15</sup&gt; N metabólico Etiquetado, inmunoprecipitación, cuantitativa Espectrometría de Masas y Modulación Affinity

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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.

De alto rendimiento de purificación de afinidad de proteínas marcadas con recombinantes

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.

GST-Su purificación: A dos pasos Affinity Protocolo Purificación Cediendo largos proteínas purificadas

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

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Purificación de proteínas libre de Método de Determinación afinidad de unión por termoforesis Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...