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. Células chapeamento <ol><li> Um dia antes da transfecção, placa de 3,5 x 10 6 células HEK293T em 10 ml de Dulbecco Modified Eagle Médium (DMEM) suplementado com 10% (v / v) de soro fetal de bovino (FBS) e 1% (v / v) de penicilina / estreptomicina (10.000 U / ml estoque) por placa de 100 mm. Placa 3 - 5 100 mm pratos por experiência / condição para garantir a recuperação de proteína suficiente. NOTA: O número de placas tem que ser determinada para cada experiência / condição, que vai contar com os níveis de expressão e a solubilidade do complexo proteína de interesse. Alternativamente, se a purificação é necessária maior escala, as linhas celulares podem ser adaptadas para crescer em suspensão em frascos rotativos de 2, mas este método não funciona para transfecções transientes. </li></ol> 2. Indução de transfecção de células ou linhas celulares estáveis <ol><li> No dia seguinte, verifique as células ao microscópio. As células devem ser 70 - 80% confluentes antes de proceeding. </li><li> Transfectar as células com uma mistura total de 7 ug de ADN de plasmídeo e 21 uL de reagente de transfecção por placa de 100 mm. Transfectar as células com um vector que expressa GFP (Tabela 1) como um controlo para a eficiência da transfecção. NOTA: Se for utilizada uma linha celular estável semelhante HEK293-T-REX ou que induz a expressão de dois ou mais complexos subunidades em resposta à doxiciclina 14, 15, o indutor terá de ser adicionado às células e incubado durante 48 h. Do mesmo modo, uma linha celular estável que induz a expressão de GFP após a adição do indutor também seria necessária para fins de validação. Avance para o passo 2.7. </li><li> Preparar a mistura de transfecção por placa de 100 mm. Num tubo de microcentrífuga de 1,5 ml, misturar 500 ul de DMEM suplementada com 7 ug de ADN plasmídeo total (correspondendo a dois ou mais plasmídeos). Num tubo de microcentrífuga separado, misturar 500 ul de DMEM sem suplementos with 21 ul de reagente de transfecção. Repita para cada reacção de transfecção. </li><li> Pipetar a solução de reagente de transfecção para a solução de DNA de plasmídeo (não misturar soluções na ordem inversa). Misturar por pipetagem de pelo menos 4 vezes. </li><li> Incubar esta mistura à temperatura ambiente durante 10 - 15 minutos, mas não mais do que 15 min. </li><li> Inclinar a placa para criar um reservatório de meios de comunicação e cuidadosamente pipetar a mistura de transfecção gota a gota, sobre o lado interior da placa, sem perturbar as células. Devolver o prato à sua posição original plana e homogeneizar suavemente para distribuir a mistura. </li><li> Incubar as células a 37 ° C numa incubadora de cultura de célula humidif içada com 5% de CO 2 (esta condição é a seguir referido como &quot;tecido incubadora de cultura&quot;). Substitua a mídia 5 horas após a transfecção (opcional). </li></ol> 3. A transfecção corrente ou Eficiência Indução <ol><li> Às 24 h pós-transfecção, visualizar as células sob uma gripemicroscópio orescent para verificar a eficiência de transfecção (ou indução de linha de células estável controle HEK293T-Rex expressando GFP). Incubar por mais 24 horas. </li></ol> 4. Purificação de Afinidade STREP (STREP AP) <ol><li> Recolher as células <ol><li> Em 48 horas após a transfecção, remova a mídia e adicionar 8 ml de solução salina 1x frio fosfato tamponada (PBS). Pipeta-se para baixo e para separar as células a partir da placa. </li><li> Recolha e transferir a suspensão de células para um tubo de 15 ml e centrifugar a células para baixo a 800 * g durante 4 min a 4 ° C. </li><li> Remover o sobrenadante de PBS. Repetir a rotação para baixo (800 xg durante 1 min a 4 ° C) para eliminar qualquer PBS residual. Armazenar o sedimento de células a -80 ° C para uso futuro ou siga o procedimento a seguir para continuar a purificação de proteínas. </li></ol></li><li> Lisar as células <ol><li> Ressuspender as peletes de células utilizando 5 x o volume de sedimento de células (~ 250 mL por placa) de Passive Lysis Buffer(PLB: Tris-HCl 20 pH 7,5, NaCl 150 mM, MgCl2 1,5 mM, DTT 1 mM, protease de cocktail inibidor de comprimido, 5% v / v de glicerol, e 1,0% de NP-40) e transferir a suspensão de células para uma 1,5 ml tubo de microcentrífuga. </li><li> Resumidamente vortex a suspensão e nutar durante 30 min a 4 ° C. </li><li> Girar a suspensão de células a 10.000 xg durante 10 min a 4 ° C. </li><li> Transfira os lisados ​​solúveis para um novo tubo de microcentrífuga de 1,5 ml e descartar os sedimentos celulares. Guardar a 10% do volume final do lisado solúvel como &quot;entrada STREP AP.&quot; Armazenar a 4 ° C. </li></ol></li><li> Equilibrando as contas STREP NOTA: Use STREP esferas para o primeiro passo AP! Complexos puxado para baixo com contas STREP recuperar significativamente mais proteína do que as puxou para baixo com contas de bandeira. <ol><li> Retire (N × 40 ul) + N ul da suspensão STREP talão 50% (n = número de amostras) e girar a 1.500 xg durante 2 min a 4 ° C. NOTA: Use de toda a mdicas outhed a esferas de pipeta. </li><li> Remover o sobrenadante e adicionar 500 mL de PLB às pérolas. Nutar a mistura grânulos durante 5 min a 4 ° C. Girar a 1500 xg durante 2 min a 4 ° C. <ol><li> Repetir para um total de 3 lavagens. Ressuspender as esferas em PLB para um volume final de 40 ul N ×. </li></ol></li><li> Adicionar 40 ul de 50% de pasta STREP talão para cada amostra de ligado celular e nutar durante 2 horas a 4 ° C. </li></ol></li><li> STREP AP <ol><li> Spin down solução a 1.500 g durante 2 min a 4 ° C. </li><li> Remover o sobrenadante e armazenar como &quot;STREP-AP por meio de fluxo (FT)&quot; a 4 ° C. </li><li> Adicionar 500 ul de Tampão de Lavagem STREP AP I (Tris-HCl 20 mM pH 7,5, NaCl 250 mM, MgCl2 1,5 mM, DTT 1 mM, 5% de glicerol e 0,2% de NP-40) para os grânulos. Nutar a mistura grânulos durante 5 min a 4 ° C e centrifugação a 1500 xg durante 2 min a 4 ° C. Descartar o sobrenadante. <ol><li> Repetir para um total de 4 lavagens. <br/&gt; NOTA: Antes da quarta lavagem, transferir a solução de grânulos para um novo tubo de microcentrifugação de 1,5 ml para reduzir o fundo de proteína. Este passo é essencial. </li></ol></li><li> Adicionar 500 ul de tampão de lavagem II (100 mM Tris-HCl pH 8,0, NaCl 150 mM, e EDTA 1 mM) e equilibrar as contas por inversão dos tubos. Girar a 1500 xg durante 2 min a 4 ° C e desprezar o sobrenadante. </li><li> Eluir a proteína de interesse com 50 ul de tampão de eluição STREP (Tris-HCl 100 mM pH 8,0, NaCl 150 mM, EDTA 1 mM, destiobiotina 2,5 mM e). Vortex a 800 rpm durante 15 min a 4 ° C. </li><li> Girar as esferas a 1.500 xg durante 1 min a 4 ° C. Recolhe-se o sobrenadante. Esta fracção corresponde à amostra &quot;A eluição STREP AP&quot;. </li><li> Guardar a fracção de esferas, a 4 ° C durante uma segunda eluição, que pode produzir-se a metade da quantidade de proteína obtida com a primeira eluição. Alíquota de 10% do AP STREP eluição por coloração com prata e / ou transferência de western16. </li></ol></li><li> Equilibrando as contas BANDEIRA <ol><li> Retire (nx 16 ul) + n ul de solução de esferas de FLAG (n = número de amostras) e girar a 1.500 xg durante 2 min a 4 ° C. NOTA: Use dicas de boca larga a contas pipeta. </li><li> Remover o sobrenadante e adicionar 500 mL de tampão de lavagem FLAG (100 mM Tris-HCl pH 8,0, 150 mM de NaCl, 1 mM de EDTA, e 0,1% de NP-40) para os grânulos. Girar a 1500 xg durante 2 min a 4 ° C. Repetir para um total de 3 lavagens. </li><li> Ressuspender as pérolas em tampão de lavagem FLAG para um volume final de 16 ul NX. </li><li> Adicionar 16 ul da suspensão FLAG grânulo para o restante eluição STREP AP e durante a noite a 4 ° nutar C. </li></ol></li></ol> 5. FLAG IP <ol><li> Spin down a suspensão esferas BANDEIRA a 1.500 g durante 2 min a 4 ° C. Salve o sobrenadante e armazenar como a &quot;bandeira IP Flow-through&quot; (FT) a 4 ° C. </li><li> Adicionar 50081; l de tampão de lavagem FLAG para a solução. Nutar durante 5 min a 4 ° C e centrifugação a 1500 xg durante 2 min a 4 ° C. Repetir para um total de 4 lavagens e remover o sobrenadante depois de cada lavagem. NOTA: Antes da quarta lavagem, transferir a solução de proteína de grânulos para um novo tubo de microcentrifugação de 1,5 ml para reduzir o fundo. Este passo é essencial. </li><li> Retirar o sobrenadante do centrifugação final e adicionar 30 uL de tampão de lavagem FLAG contendo 200 ng / mL de péptido FLAG nas pérolas. Vortex a 800 rpm durante 2 horas a 4 ° C. </li><li> Girar a suspensão a 1500 xg durante 2 min a 4 ° C. Colher o sobrenadante e armazenar a 4 ° C como o &quot;BANDEIRA IP eluição.&quot; NOTA: As amostras de eluição pode ser confirmada por coloração de prata e / ou transferência de Western utilizando protocolos padrão de 16, e está pronto para mais ensaios de proteína. Ao configurar um western blot, execute todas as seguintes amostras: (1) STREP AP entrada, (2) STREP AP FT, (3) STREP AP Elution, (4) BANDEIRA IP FT, e (5) BANDEIRA IP eluição. Volumes carregados terá de ser determinado para cada experimento. Todas as amostras recolhidas e contas podem ser armazenadas a 4 ° C para armazenagem a curto prazo e a -80 ° C para armazenamento a longo prazo. Amostras de TAP podem ser analisados por espectrometria de massa para verificar a identidade dos seus componentes, para identificar novas modificações pós-traducionais (PTMs), e / ou para descobrir qualquer interacção nova proteína com o complexo purificado 2, 17. Para este efeito, após a execução de um gel de mancha de Coomassie ou prata, as bandas podem ser excisadas a partir do gel utilizando um bisturi limpa. Vários excelentes comentários que discutem métodos de espectrometria de massa foram publicados anteriormente 18, 19. </li></ol>

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The authors have nothing to disclose.

O protocolo aqui descrito para a expressão e o isolamento de complexos de proteínas a partir de células eucarióticas não se limita à caracterização bioquímica de tais conjuntos moleculares, mas também pode ser utilizado para a identificação de novos interactores e modificações pós-traducionais que poderia regular a sua função. A utilização de etiquetas de afinidade não se restringe ao que é mencionado neste protocolo; mas a experiência sugere que o uso de STREP como o primeiro passo AP aumenta sign...

<html> Interações proteína-proteína (IBP) são fundamentais para a regulação precisa de processos biológicos 1, e mais estudos sobre estas PPIs poder informar sobre a sua função 2. Várias abordagens têm sido desenvolvidos para o estudo e caracterização de IPP, bem como para a identificação de novo de novos componentes de proteínas reguladoras. Em 1989, Stanley Campos e colegas relataram a levedura de dois híbridos (Y2H) ensaio 3. Esta abordagem permite a identificação imparcial e abrangente de interactianos (de caçar) para uma proteína definida de interesse (isca) em Saccharomyces cerevisiae. Para além da sua utilidade notável para a descoberta de IPP, o ensaio Y2H pode ser utilizado para caracterizar pares de proteínas em células de levedura, que define o mínimo de domínios interactuantes, e a identificação de mutações que abolem essas interacções. Ao modificar o ensaio Y2H, IPP também pode ser estudado em células de mamíferoclass = &quot;xref&quot;&gt; 4. Variações do ensaio Y2H (por exemplo, sistema de levedura de três híbridos) também pode ser aplicada ao estudo de proteínas e de ARN-ligante orgânico proteína-pequeno em células. Outra ferramenta utilizada para estudar os IPP num sistema homólogo é o ensaio de co-imunoprecipitação (co-IP) 5. Ao utilizar um anticorpo para imunoprecipitar uma proteína de interesse, o ensaio de co-IP permite aos investigadores monitorar IPP em células para diferentes condições ambientais e experimentais situações. A utilização de proteínas marcadas no epitopo (por exemplo, FLAG, Myc, estreptococos e HA, entre outros), a purificação por afinidade (AP) métodos facilitou o isolamento de proteínas a partir de misturas complexas de proteínas para vários ensaios a jusante, incluindo Western blot, coloração de prata , e análise enzimática. No entanto, nenhuma destas abordagens anteriores permitem o isolamento de grandes quantidades de complexos de proteínas para caracterização adicional in vitro incluindo umssays, descoberta de subunidades reguladoras por espectrometria de massa, e a identificação de modificações pós-traducionais. Uma versão melhorada do método de AP AP é chamado tandem (TAP), que é uma técnica de purificação para o estudo de IPP através da criação de uma proteína de fusão com dois epitopos que se purifica por meio de dois pontos de acesso subsequentes 6, 7. Neste artigo, apresentamos uma variação do método TAP para complexos de proteína de purificação em que duas subunidades são marcadas com epítopos diferentes e depois purificado através de dois APs sequenciais (STREP AP seguido por FLAG IP). Em primeiro lugar, proporcionar uma visão minimalista da TAP (Figura 1) e, em seguida, uma descrição detalhada de todas as etapas experimentais (Figura 2), de modo que os investigadores possam aplicá-las a seu complexo de proteína de interesse. Para demonstrar a aplicabilidade do método de TAP, nós escolhemos um bem caracterizado ciclina-CDK complexa (referido como PTEFB quinase), que é composta da subunidade reguladora da ciclina T1 (CycT1) e uma cinase (CDK9), e está envolvido na regulação da transcrição por ARN-polimerase II (pol II) 8, 9, 10. P-TEFb fosforila o domínio C-terminal de pol II e os seus associados factores de alongamento negativos, o que alivia a pausa da transcrição no promotor e facilita assim a transcrição de alongamento 11, 12, 13. Com essa interação conhecida em mente, STREP-marcado CycT1 e CDK9 marcado com FLAG foram mais expressos em células HEK293T. Uma experiência recíproca TAP foi realizada com CDK9 marcaram-STREP e marcado com FLAG CycT1 para validar ainda mais que a interacção da proteína é independente dos epítopos utilizados. As células foram recolhidas e lisadas 48 h após a transfecção. O ligado solúvel foi purificado por TAP (STREP AP seguido por FLAGIP). Entrada e proteínas purificadas foram analisadas por Western blot e por coloração com prata (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

A purificação dos complexos de ácido nucleico de proteína-proteína-proteína activa e é essencial para a caracterização das actividades enzimáticas e de identificação de novo de novas subunidades e as modificações pós-traducionais. sistemas bacterianos para permitir a expressão e purificação de uma grande variedade de polipéptidos simples e complexos de proteínas. No entanto, este sistema não permite a purificação de subunidades proteicas que contêm modificações pós-translacionais (por exemplo, fosforilação e acetilação), e a identificação de novas subunidades reguladoras que estão apenas presentes / expressas no sistema eucariota. Aqui, nós fornecemos uma descrição detalhada de um romance, robusta e eficiente método de purificação de afinidade em tandem (TAP), utilizando Streptomyces e proteínas marcada com Flag que facilita a purificação de complexos de proteínas com proteínas marcadas no epitopo transiente ou estavelmente expresso a partir de células eucarióticas. Este protocolo pode ser aplicado para caracterizar Protein funcionalidade complexa, para descobrir modificações pós-translacionais sobre subunidades complexas, e para identificar novos componentes complexos de regulação por espectrometria de massa. Notavelmente, este método TAP pode ser aplicado para estudar complexos proteicos formados por componentes eucarióticas ou patogénicos (viral e bacteriana), obtendo-se assim uma grande variedade de oportunidades experimentais jusante. Propomos que os investigadores que trabalham com complexos de proteína poderia utilizar esta abordagem de muitas maneiras diferentes.

Neste artigo, demonstrar a aplicabilidade do método de TAP para o bem caracterizado CycT1-CDK9 complexo (também conhecida como P-TEFb quinase). Os plasmídeos que codificam ciclina T1-STREP (CycT1: S) e CDK9-FLAG (CDK9: F), ou CDK9-STREP (CDK9: S) e ciclina T1-FLAG (CycT1: F) (Tabela 1), foram transfectados em células HEK293T . Os controlos negativos incluíram as transfecções com um vector vazio e o CDK9...

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.

A purificação de afinidade em tandem de complexos de proteínas a partir de células eucarióticas

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 visualizações.  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.

Vídeo: 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.

Proteína de captura por afinidade complexos a partir de células de mamíferos Cryomilled

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

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 purificada por afinidade complexos de proteínas por Azul Native PAGE e Proteína Correlação Profiling

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 em 1: uma etapa de purificação da afinidade para a análise paralela de proteína-proteína e proteína-metabólito complexos

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.

Purificação da afinidade do cloroplasto Translocon proteínas complexos usando a marca de torneira

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.

Identificação de parceiros de proteína Interagir utilizando Tandem purificação por afinidade

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

Biologia

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.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

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.

A Plataforma de Produção de Proteína Complexo MultiBac no 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.

Identificando interacção proteína-proteína em<em&gt; Drosophila</em&gt; Chefes Adulto por Tandem Affinity Purification (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.

Purificação de Native Complexos de estudo estrutural usando um 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.

Ensaio de purificação de afinidade em tandem para estudar interações proteína-proteína

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

A purificação de afinidade em tandem de complexos de proteínas a partir de células eucarióticas

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