Name: tandem affinity purification of protein complexes from eukaryotic cells
Uploaded: 2017-01-26T14:00:00.000+00:00
Duration: 11 min 30 s
Description: Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under award number R01AI114362 and Welch Foundation grant I-1782 to Iv&#225;n D'Orso.

1. Las células Plating <ol><li> Un día antes de la transfección, la placa de 3,5 x 10 6 células HEK293T en 10 ml de de Eagle modificado por Dulbecco (DMEM) suplementado con 10% (v / v) de suero bovino fetal (FBS) y 1% (v / v) de penicilina / estreptomicina (10.000 U / ml de solución madre) por placa de 100 mm. Plate 3 - 5 100 mm platos por experimento / condición para asegurar una recuperación suficiente proteína. NOTA: El número de placas tiene que ser determinada para cada experimento / condición, que se basará en los niveles de expresión y la solubilidad del complejo de proteína de interés. Alternativamente, si se necesita la purificación a gran escala, las líneas celulares pueden ser adaptadas para crecer en suspensión en matraces de agitación 2, pero este método no funcionará para transfecciones transitorias. </li></ol> 2. Inducir la transfección de células o líneas celulares estables <ol><li> Al día siguiente, comprobar las células bajo el microscopio. Las células deben ser de 70 - 80% de confluencia antes de proceeding. </li><li> Transfectar las células con una mezcla total de 7 g de ADN plásmido y 21 l de reactivo de transfección por plato 100 mm. Células transfectar con un vector que expresa GFP (Tabla 1) como control para la eficiencia de transfección. NOTA: Si se utiliza una línea celular estable similares HEK293-T-REx o que induce la expresión de dos o más complejos de subunidades en respuesta a la doxiciclina 14, 15, el inductor tendrá que ser añadido a las células y se incubaron durante 48 hr. Del mismo modo, una línea celular estable que induce la expresión de GFP después de la adición del inductor también se requiere para fines de validación. Continúe en el paso 2.7. </li><li> Preparar la mezcla de transfección por placa de 100 mm. En un tubo de microcentrífuga de 1,5 ml, mezclar 500 l de DMEM sin suplementos con 7 g de ADN de plásmido total (correspondiente a dos o más plásmidos). En un tubo de microcentrífuga separado, mezclar 500 l de DMEM sin suplementos with 21 l de reactivo de transfección. Repita este procedimiento para cada reacción de transfección. </li><li> Pipetear la solución del reactivo de transfección en la solución de ADN del plásmido (no se mezclan soluciones en el orden inverso). Mezclar pipeteando al menos 4 veces. </li><li> Incubar la mezcla a temperatura ambiente durante 10 a 15 min, pero no más de 15 min. </li><li> Inclinar la placa para crear un depósito de medio y cuidadosamente pipetear la mezcla de transfección gota a gota sobre el lado interior de la placa sin perturbar las células. Devolver el plato a su posición plana original y girar suavemente para distribuir la mezcla. </li><li> Se incuban las células a 37 ° C en una incubadora de cultivo de células humidificado con 5% de CO 2 (esta condición se denomina en adelante como &quot;incubadora de cultivo de tejidos&quot;). Reemplazar los medios de comunicación 5 horas después de la transfección (opcional). </li></ol> 3. La transfección de cheques o la eficiencia de inducción <ol><li> A las 24 h después de la transfección, visualizar las células bajo una gripeorescent microscopio para comprobar la eficiencia de transfección (o la inducción del control HEK293T-Rex línea celular estable que expresa GFP). Se incuba durante otras 24 horas. </li></ol> 4. STREP Purificación por Afinidad (STREP AP) <ol><li> Recoger las células <ol><li> A las 48 horas después de la transfección, saque la tarjeta y añadir 8 ml de solución salina tamponada con fosfato 1x frío (PBS). Pipetear arriba y abajo para separar las células de la placa. </li><li> Recoger y transferir la suspensión de células en un tubo de 15 ml y centrifugar las células hacia abajo a 800 xg durante 4 minutos a 4 ° C. </li><li> Eliminar el sobrenadante de PBS. Repetir el giro hacia abajo (800 xg durante 1 min a 4 ° C) para eliminar cualquier PBS residual. Almacenar el sedimento celular a -80 ° C para su uso futuro o seguir el procedimiento siguiente para continuar la purificación de proteínas. </li></ol></li><li> Lisar las células <ol><li> Volver a suspender los sedimentos celulares utilizando 5 x el volumen de sedimento celular (~ 250 l por placa) de tampón de lisis pasiva(PLB: 20 mM Tris-HCl pH 7,5, NaCl 150 mM, 1,5 mM MgCl 2 mM, DTT 1, proteasa tableta de cóctel inhibidor, 5% v / v de glicerol, y 1,0% NP-40) y transferir la suspensión celular a una 1,5 ml tubo de microcentrífuga. </li><li> Brevemente vórtex con la suspensión y inclinar la cabeza durante 30 minutos a 4 ° C. </li><li> Centrifugar la suspensión de células a 10.000 xg durante 10 min a 4 ° C. </li><li> La transferencia de los lisados ​​solubles en un nuevo tubo de microcentrífuga de 1,5 ml y desechar los sedimentos celulares. Ahorra un 10% del volumen final del lisado soluble como &quot;Input STREP AP.&quot; Almacenar a 4 ° C. </li></ol></li><li> Equilibrar las cuentas STREP NOTA: Los granos Uso STREP para la primera etapa AP! Complejos tira hacia abajo con los granos STREP recuperan significativamente más proteína que los derribado con perlas de bandera. <ol><li> Sacar (n × 40 l) n + l de la suspensión de cuentas STREP 50% (n = número de muestras) y centrifugar a 1500 xg durante 2 min a 4 ° C. NOTA: Use-m de anchoconsejos outhed a perlas de pipeta. </li><li> Eliminar el sobrenadante y añadir 500 l de PLB a las perlas. Inclinar la cabeza la mezcla de los granos durante 5 min a 4 ° C. Centrifugar a 1500 xg durante 2 min a 4 ° C. <ol><li> Repita para un total de 3 lavados. Resuspender las perlas en PLB a un volumen final de n × 40 l. </li></ol></li><li> Añadir 40 l de 50% de suspensión STREP de cuentas para cada muestra de lisado celular y inclinar la cabeza durante 2 horas a 4 ° C. </li></ol></li><li> STREP AP <ol><li> Centrifugar la solución a 1.500 xg durante 2 min a 4 ° C. </li><li> Eliminar el sobrenadante y almacenar como &quot;STREP AP de flujo continuo (FT)&quot; a 4 ° C. </li><li> Añadir 500 l de STREP AP tampón de lavado I (Tris-HCl 20 mM pH 7,5, NaCl 250 mM, 1,5 mM MgCl 2 mM, DTT 1, 5% de glicerol y 0,2% NP-40) a las perlas. Inclinar la cabeza la mezcla de perlas durante 5 min a 4 ° C y se centrifuga a 1500 xg durante 2 min a 4 ° C. Descartar el sobrenadante. <ol><li> Repita para un total de 4 lavados. <br/&gt; NOTA: Antes del cuarto lavado, transferir la solución de perlas a un nuevo tubo de microcentrífuga de 1,5 ml a reducir el fondo de proteínas. Este paso es esencial. </li></ol></li><li> Añadir 500 l de tampón de lavado II (100 mM Tris-HCl pH 8,0, NaCl 150 mM, y EDTA 1 mM) y equilibrar las cuentas por inversión de los tubos. Centrifugar a 1500 xg durante 2 min a 4 ° C y descartar el sobrenadante. </li><li> Eluir la proteína de interés con 50 l de STREP Tampón de Elución (Tris-HCl 100 mM pH 8,0, NaCl 150 mM, EDTA 1 mM, y destiobiotina 2,5 mM). Vortex a 800 rpm durante 15 min a 4 ° C. </li><li> Centrifugar las perlas a 1.500 xg durante 1 min a 4 ° C. Recoger el sobrenadante. Esta fracción corresponde a la muestra &quot;STREP AP elución&quot;. </li><li> Guardar la fracción perlas a 4 ° C para una segunda elución, lo que podría producir hasta la mitad de la cantidad de proteína obtenida con la primera elución. Alícuota de 10% de la STREP AP elución para la tinción de plata y / o el Western Blot16. </li></ol></li><li> Equilibrar las cuentas BANDERA <ol><li> Sacar (nx 16 l) n + l de solución de perlas de FLAG (n = número de muestras) y centrifugar a 1500 xg durante 2 min a 4 ° C. NOTA: Utilice consejos de boca ancha a perlas de pipeta. </li><li> Eliminar el sobrenadante y añadir 500 l de tampón de lavado FLAG (100 mM Tris-HCl pH 8,0, NaCl 150 mM, EDTA 1 mM, y 0,1% NP-40) a las perlas. Centrifugar a 1500 xg durante 2 min a 4 ° C. Repita para un total de 3 lavados. </li><li> Resuspender las perlas en tampón de lavado FLAG a un volumen final de 16 l nx. </li><li> Añadir 16 l de la suspensión de la BANDERA de talón al resto de elución STREP AP y alojamiento inclinar la cabeza a 4 ° C. </li></ol></li></ol> 5. BANDERA IP <ol><li> Centrifugar la suspensión perlas FLAG a 1.500 xg durante 2 min a 4 ° C. Guardar el sobrenadante y tienda como la &quot;bandera IP de flujo continuo&quot; (FT) a 4 ° C. </li><li> Añadir 50081; l de tampón de lavado FLAG a la solución. Inclinar la cabeza durante 5 min a 4 ° C y se centrifuga a 1500 xg durante 2 min a 4 ° C. Repita para un total de 4 lavados y eliminar el sobrenadante después de cada lavado. NOTA: Antes de que el cuarto lavado, transferir la solución de proteína-cuentas a un nuevo tubo de microcentrífuga de 1,5 ml para reducir el fondo. Este paso es esencial. </li><li> Eliminar el sobrenadante del centrifugado final y añadir 30 l de tampón de lavado que contiene FLAG 200 ng / l de péptido FLAG en las perlas. Vórtice a 800 rpm durante 2 horas a 4 ° C. </li><li> Centrifugar la suspensión a 1.500 xg durante 2 min a 4 ° C. Se recoge el sobrenadante y se almacena a 4 ° C como la &quot;bandera IP elución.&quot; NOTA: Las muestras de elución se pueden confirmar por tinción de plata y / o de transferencia de Western usando protocolos estándar 16, y están listos para más ensayos de proteínas. Al configurar una transferencia de western, ejecutar todas las siguientes muestras: (1) PEIF AP de entrada, (2) STREP AP FT, (3) STREP AP Elutien, (4) BANDERA IP FT, y (5) BANDERA IP elución. Los volúmenes cargados tendrán que ser determinado para cada experimento. Todas las muestras recogidas y los granos se pueden almacenar a 4 ° C para el almacenamiento a corto plazo y a -80 ° C para el almacenamiento a largo plazo. Muestras TAP podrían ser analizados mediante espectrometría de masas para verificar la identidad de sus componentes, para identificar nuevos modificaciones post-traduccionales (PTMs), y / o para descubrir cualquier interacción nueva proteína con el complejo purificado 2, 17. Para este propósito, después de ejecutar una mancha de gel de Coomassie o plata, las bandas pueden ser extirpados del gel utilizando un escalpelo limpio. Varios excelentes críticas que discuten métodos de espectrometría de masas se han publicado 18, 19. </li></ol>

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The authors have nothing to disclose.

El protocolo descrito aquí para la expresión y el aislamiento de los complejos de proteínas a partir de células eucariotas no se limita a la caracterización bioquímica de tales conjuntos moleculares, pero también puede ser utilizado para la identificación de nuevos interactores y modificaciones posteriores a la traducción que podría regular su función. La utilización de etiquetas de afinidad no se limita a lo que se menciona en este protocolo; pero nuestra experiencia sugiere que el uso de STREP como el prim...

<html> Interacciones proteína-proteína (IBP) son fundamentales para la regulación precisa de los procesos biológicos 1, y más estudios sobre estos IBP puede informar acerca de su función 2. Varios enfoques se han ideado para el estudio y caracterización de los IBP, así como para la identificación de novo de nuevos componentes de la proteína de regulación. En 1989, Stanley Fields y sus colegas informaron de la levadura de dos híbridos (Y2H) de ensayo 3. Este enfoque permite la identificación imparcial y exhaustiva de interactores (presas) para una proteína de interés definida (cebo) en Saccharomyces cerevisiae. Además de su notable utilidad para el descubrimiento de los IBP, el ensayo Y2H se puede utilizar para la caracterización de pares de proteínas en células de levadura, la definición de dominios que interactúan mínimas, y la identificación de mutaciones que suprimen tales interacciones. Modificando el ensayo Y2H, IBP también puede estudiarse en células de mamíferoclass = &quot;xref&quot;&gt; 4. Las variaciones del ensayo de Y2H (por ejemplo, sistema de levadura de tres híbridos) también se pueden aplicar para estudiar la proteína-ARN y las interacciones ligando orgánico de proteínas pequeñas en las células. Otra herramienta usada comúnmente para estudiar los IBP en un sistema homólogo es la co-inmunoprecipitación (co-IP) de ensayo 5. Mediante el uso de un anticuerpo para inmunoprecipitar una proteína de interés, el ensayo de co-IP permite a los investigadores monitorizar los IBP en las células para diferentes condiciones ambientales y situaciones experimentales. El uso de proteínas etiquetadas con epítopo (por ejemplo, FLAG, Myc, STREP, y HA, entre otros) en la purificación por afinidad (AP) métodos ha facilitado el aislamiento de proteínas a partir de mezclas complejas de proteínas de varios ensayos de aguas abajo, incluyendo transferencia de Western, tinción de plata , y análisis enzimático. Sin embargo, ninguno de estos enfoques anteriores permiten el aislamiento de grandes cantidades de complejos de proteínas para una caracterización adicional incluyendo in vitro unassays, descubrimiento de subunidades reguladoras por espectrometría de masas, y la identificación de las modificaciones post-traduccionales. Una versión mejorada del método de AP se llama Tandem AP (TAP), que es una técnica de purificación para el estudio de los IBP mediante la creación de una proteína de fusión con dos epítopos que se purifica a través de dos puntos de acceso posteriores 6, 7. En este artículo, se presenta una variación del método de TAP complejos de proteínas de purificación en el que dos subunidades etiquetados con diferentes epítopos y después se purificó a través de dos puntos de acceso secuencial (STREP AP seguido de FLAG IP). En primer lugar, proporcionar una visión minimalista de TAP (Figura 1) y, a continuación una descripción detallada de todos los pasos experimentales (Figura 2), por lo que los investigadores puedan aplicarlas a su complejo de proteína de interés. Para demostrar la aplicabilidad del método TAP, elegimos un bien caracterizado ciclina-CDK complejo (denominado PTEFB quinasa), que se compone de la ciclina T1 subunidad reguladora (CycT1) y una quinasa (CDK9), y está implicado en la regulación de la transcripción por la ARN polimerasa II (Pol II) 8, 9, 10. P-TEFb fosforila el dominio C-terminal de Pol II y sus asociados factores de elongación negativos, lo que alivia la pausa de la transcripción en el promotor y de ese modo facilita la elongación de la transcripción 11, 12, 13. Con esta interacción conocida en mente, Strep-etiquetados CycT1 y CDK9 marcado con FLAG fueron sobreexpresado en células HEK293T. Un experimento recíproco TAP se realizó con CDK9 Strep-etiquetados y la bandera de etiquetado CycT1 para validar además que la interacción de proteínas es independiente de los epítopos utilizados. Las células se recogieron y se lisaron 48 h después de la transfección. El lisado soluble se purificó por TAP (STREP AP seguido por FLAGIP). Entrada y proteínas purificadas se analizaron por Western blot y tinción de plata (Figura 3).

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH3002.2FS</td><td></td></tr><tr><td>Fetal bovine serum</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH30071</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>MP091670049</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen Laboratories</td><td>SL100688</td><td></td></tr><tr><td>Protease inhibitor cocktail&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>11836153001</td><td></td></tr><tr><td>STREP-Tactin Superflow</td><td>IBA Lifesciences</td><td>2-1208-010</td><td></td></tr><tr><td>STREP-tag elution buffer</td><td>IBA Lifesciences</td><td>2-1000-025</td><td></td></tr><tr><td>EZview Red ANTI-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>F2426</td><td></td></tr><tr><td>Corning 100 mm &amp;times; 20 mm style dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td><td>Corning&amp;nbsp;</td><td>430167</td><td></td></tr><tr><td>Protein Lo-Bind Eppendorf</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific&amp;nbsp;</td><td>02-215-370</td><td></td></tr><tr><td>48 hole micro tube foam rack</td><td>Fisher Scientific</td><td>02-215-386</td><td></td></tr><tr><td>Labquake shaker rotisserie&amp;nbsp;</td><td>Thermo&amp;nbsp;</td><td>415110</td><td></td></tr></tbody></table>

tandem affinity purification of protein complexes from eukaryotic cells

La purificación de activo proteína-proteína y complejos de proteína-ácido nucleico es crucial para la caracterización de las actividades enzimáticas y de novo identificación de nuevas subunidades y modificaciones post-traduccionales. sistemas bacterianos permiten la expresión y purificación de una amplia variedad de polipéptidos individuales y complejos de proteínas. Sin embargo, este sistema no permite la purificación de subunidades de proteínas que contienen modificaciones post-traduccionales (por ejemplo, la fosforilación y la acetilación), y la identificación de nuevas subunidades reguladoras que sólo están presentes / expresadas en el sistema eucariota. Aquí, proporcionamos una descripción detallada de una novela, robusto y eficiente método de purificación por afinidad en tándem (TAP) usando estreptomicina y proteínas bandera de etiquetado que facilita la purificación de complejos de proteínas con proteínas de forma transitoria o estable expresado etiquetadas con epítopo de las células eucariotas. Este protocolo se puede aplicar a caracterizar protein funcionalidad compleja, para descubrir las modificaciones posteriores a la traducción en subunidades complejos, e identificar nuevos componentes complejos de regulación mediante espectrometría de masas. En particular, este método TAP se puede aplicar para estudiar complejos de proteínas formadas por componentes eucariotas o patógenos (virales y bacterianas), produciendo de este modo una amplia gama de oportunidades experimentales aguas abajo. Proponemos que los investigadores que trabajan con complejos de proteínas podrían utilizar este enfoque de muchas maneras diferentes.

En este artículo, se demuestra la aplicabilidad del método TAP para el bien caracterizado CycT1-CDK9 compleja (también conocido como P-TEFb quinasa). Los plásmidos que codifican la ciclina T1-STREP (CycT1: S) y CDK9-FLAG (CDK9: F), o CDK9-STREP (CDK9: S) y Ciclina T1-FLAG (CycT1: F) (Tabla 1), se transfectaron en células HEK293T . Los controles negativos incluyeron transfecciones con un vector vacío y la...

Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Tandem Affinity purificación de complejos de proteínas a partir de células eucariotas

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

tandem-affinity-purification-protein-complexes-from-eukaryotic

Research

JoVE Journal

Biochemistry

14.9K vistas.  The University of Texas Southwestern Medical Center. We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Video: Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Complejo de proteínas de captura por afinidad a partir de células de mamífero Cryomilled

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Investigación

Bioquímica

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolver purificado por afinidad Complejos de Proteína de Blue nativo PAGE y de proteínas de perfiles de correlación

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 en 1: purificación de la afinidad para el análisis paralelo de proteínas y complejos de proteína-metabolito de un solo paso

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Purificación de la afinidad del cloroplasto Translocon complejos utilizando la etiqueta de grifo

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

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.

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 ...

Biología

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 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

El Protein Complex Plataforma de Producción MultiBac en el EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

La identificación de la interacción proteína-proteína en<em&gt; Drosophila</em&gt; Jefes adultas por Tandem Affinity purificación (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Purificación de Complejos nativo para el estudio estructural se utiliza un método Tag Tandem Affinity

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Ensayo de purificación de afinidad en tándem para estudiar las interacciones proteína-proteína

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

Tandem Affinity purificación de complejos de proteínas a partir de células eucariotas

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