Agradecemos a Anne-Marie Dion-Cote para discussões que levaram ao desenvolvimento do método. JK e RB são FQNRT estudiosos de doutorado, M.-MG é um estudioso Vanier CIHR e J.-YM é pesquisador sênior FRSQ. Este trabalho foi apoiado por fundos de Ciências Naturais e do Conselho de Pesquisa de Engenharia de JY. M.

1. Produção de recombinante de baculovírus Os baculovírus são gerados utilizando o Sistema de Expressão de Baculovirus Bac-to-Bac de Invitrogen, principalmente de acordo com o protocolo do fabricante, com apenas ligeiras modificações: <ol><li> Um vetor pFastBac1 modificado (Gibco, a vida) foi criado contendo o GST e Seus-tags (Figura 2). O local de clonagem múltipla (MCS) de pET-52b (+) vector (Novagen), que contém uma etiqueta 10xHis, foi inserido no mcs de um vector pFastBac1 para gerar pFastBac1-Strep-10xHis. A sequência de MCS pET-52b (+) foi amplificado por PCR entre os locais de restrição Xbal e BlpI, a adição de um local de restrição de BglII no terminal 5 &#39;da extremidade e um local de restrição Hind III na extremidade 3&#39;. O produto de PCR foi então clonado entre BamHI e HindIII sítio de restrição de pFastBac1, usando a compatibilidade de sequências de restrição de BamHI e BglII. Uma vez que o objetivo era gerar um pFastBac1 permitindo a expressão deproteína recombinante com estreptavidina-e 10xHis-tags, o sítio de restrição BamHI de pET-52b (+) sequência de MCS não foi utilizado. Além disso, o sítio de restrição XbaI não foi usado para evitar a redundância de sítios de restrição que ocorrem entre os sites que já estão no MCS de pFastBac1 e estes inserido com o MCS de pET-52b (+). A sequência de codificação de GST com o local de reconhecimento PreScission Protease (LeuGluValLeuPheGln / GlyPro) foi então inserido no pFastBac1-Strep-10xHis. A sequência de cDNA a partir de GST pGEX-6P-2 (GE Healthcare) foi amplificado por PCR com iniciadores que contêm sítios de restrição Ncol e Kpnl na extremidade 5 &#39;e 3&#39; das extremidades, respectivamente. O local Ncol permite a adição de um codão de iniciação, mas também uma alanina para o GST. O produto de PCR foi então clonado no pFastBac1-Strep-10xHis entre os sítios de restrição Ncol e Kpnl, resultando na deleção da etiqueta de estreptavidina. O novo vetor de fusão assim obtido foi denominado pFastBac1-GST-10xHis. </li><li> Clonar o cDNA codificante para a protein de interesse no vector pFastBac-GST-10xHis entre a GST (N-terminal) e a cauda de His (C-terminal). Ter cuidado para remover o codão de terminação para permitir que o cDNA de fusão com o His-tag no terminal C. </li><li> Transforme E. coli DH10Bac com o pFastBac recombinante (obtida no passo 1.2) para gerar o bacmid. Permitir que as colónias crescer durante a noite a 37 ° C e várias horas a 30 ° C em placas de selecção contendo Bluo-gal e IPTG (10 mg / ml de tetraciclina, 50 ug / ml de canamicina, 7 ug / ml de gentamicina, 100 ug / ml de Bluo- Gal, 40 ug / ml de IPTG). </li><li> Escolha e restreak duas colônias brancas (a partir do passo 1.3) em placas de seleção para confirmar o fenótipo branco. </li><li> Inocular 2 ml de LB contendo 10 ug / ml de tetraciclina, 50 ug / ml de canamicina, 10 mg / ml de gentamicina, com as duas colónias brancas a partir do passo 1.4 e deixá-los crescer durante a noite, sob agitação (250 rpm) a 37 ° C. </li><li> A fim de purificar o ADN de bacmid, sedimentar as bactérias do passo 1.5,ressuspender as células em 300 uL de solução I e adicionar 300 mL de solução II (kit Maxiprep da Qiagen). Incubar durante 5 min à temperatura ambiente, adicionar 300 mL de 3 M KOAc, pH 5,5 e incubação durante 10 min em gelo. Centrifugar as amostras a 4 ° C, a velocidade máxima durante 10 min. Adicionar 800 mL de isopropanol para os sobrenadantes e incubar por 10 min em gelo. Centrifugar as amostras durante 15 min a 4 ° C e lava-se o sedimento com 500 ul de etanol a 70%. Deixar o sedimento secar e ressuspender o sob condições estéreis, em 40 ul de Tris-EDTA a pH 8,0. O ADN de bacmid deve ser armazenada a 4 ° C. </li><li> Gerar baculovírus, conforme descrito no protocolo do fabricante. Os baculovírus pode ser armazenado a 4 ° C protegidos da luz. </li></ol> 2. Recombinant Protein Expression <ol><li> De modo a escolher o baculovírus mais eficiente para a purificação e para controlar o tempo do pico da expressão da proteína é alcançada, execute um mini-ensaio de uma infecçãos seguintes: Infect 2 x 10 7 de Sf9 infectadas células cultivadas a 27 ° C em meio de Grace (complementado com SBF a 10% e 1% P / S) com 133 ul (1/150) de baculovírus. Recolhe alíquotas de 1,5 ml a 0, 24, 48, e 72 horas pós-infecção. Sedimentar as células e analisar o nível da proteína de interesse por western blotting utilizando anticorpos anti-tag de expressão. </li><li> Use o baculovírus mais eficiente a partir do passo 2.1 para infectar um spinner contendo 1,0 x 10 6 células Sf9 por ml em 500 ml de mídia com uma proporção de 1/150 entre o vírus e meios de comunicação. Permitir que as células infectadas crescer em suspensão a 27 ° C e colheita das células, quando a expressão máxima de proteína (como determinado no passo 2.1 acima) é alcançado. As células sedimentadas podem ser armazenadas a -80 ° C até posterior utilização. </li></ol> 3. Preparação de Lisado celular solúvel Todos os seguintes passos de incubação são realizados a 4 ° C sob rotação suave. <ol><li> Lyse SAs células que expressam f9 sua proteína de interesse, em ~ 20 ml de tampão de ligação de GST (NaCl 150 mM, EDTA 1 mM, 0,05% de Triton-X-100, DTT 1 mM em PBS1X para uma concentração final de NaCl de 250 mM, e inibidores da protease cocktail (Roche). Num banho de água gelada, a solução 20x Dounce com um homogeneizador de Dounce, sonicado 3x 30 segundos cada (produção de 70%), e novamente Dounce 20x. </li><li> (Opcional). Incubar o ligado total de células com 1 mM de MgCl 2 e 7 nuclease Benzonase (2,5 U / ml) durante 30 min a 4 ° C para remover o ADN de contaminação ou de ARN. </li><li> Centrifugar a 18.000 rpm durante 30 min a 4 ° C. Manter o sobrenadante. </li><li> (Opcional). Centrífuga-se o sobrenadante de novo durante 30 min a 18.000 rpm a 4 ° C para obter um lisado solúvel claro. Passar o sobrenadante através de um filtro de 0,2 ou 0,45 mM para evitar o entupimento. </li></ol> 4. Ligação da proteína em GST Beads <ol><li> Incubar o ligado celular solúvel com 1 ml de pérolas de GST (pré-lavado duas vezes com10 ml de tampão GST) de ligação, durante 1 hora a 4 ° C, sob suave rotação. </li></ol> Comentário: O tempo de incubação deve ser optimizado de acordo com a estabilidade da proteína e eficiência de ligação. <ol start="2"><li> Rotação rápida a 700 rpm e remover o sobrenadante contendo as proteínas não ligadas (o que pode ser mantido para uma análise mais aprofundada). Lavar as proteínas ligadas a GST (Figura 3B, pista 1) com tampão de lavagem de GST (GST com tampão de ligação NaCl 350 mM final). </li><li> Repita o passo 4.2 2x. Na última lavagem, remover tanto sobrenadante quanto possível. </li><li> Incubar os grânulos com 5 mM de ATP e 15 mM de MgCl 2 (em 10 ml de tampão de ligação a GST) durante 1 hora a 4 ° C para evitar ligação não específica de proteínas de choque de calor 8. Lavar as pérolas três vezes com tampão de lavagem de GST. </li></ol> 5. PreScission clivagem do GST <ol><li> Centrifugar as esferas de GST, remover o sobrenadante e lavar as contas de GST com P5 buffer (50 NaHPO 4 mM pH 7,0, NaCl 500 mM, 10% de glicerol, 0,05% de Triton-X-100, 5 mM de imidazole). </li><li> Incubar as GST-esferas com enzimas PreScission, em tampão P5 a partir de 3 horas a durante a noite a 4 ° C. (Dividir as esferas de GST em fracções de 100 mL e adicionar 4-8 unidades (2-4 ul) de enzima diluído em 100 ul de tampão de P5). </li></ol> Comentário: O tempo de incubação do passo PreScission precisa ser optimizada de acordo com o peso molecular, assim como a estabilidade da proteína. Se se observar degradação, este tempo de incubação pode ser reduzido até um mínimo de 2 horas em vez de durante a noite. <ol start="3"><li> Rotação rápida e recolher o sobrenadante. Repita esta etapa duas vezes depois de adicionar 100 ml de P5 de tampão aos grânulos e reunir todas as fracções (Figura 3B, pista 2). </li></ol> Comentário: Os restantes grânulos pode ser usada para a análise de eficácia de clivagem (Figura 3B </strong&gt;, pista 3). Normalmente, 70-80% da proteína é clivada. 6. Para TALON metal Affinity Resin-A ligação às proteínas <ol><li> Divide-se a eluição a partir de cima em 3 fracções de 1 ml e incubar cada uma com 100 de resina de afinidade com metal Talon grânulos secos ul (pré-lavado duas vezes com tampão de P5) durante 1 hora sob rotação. </li></ol> Comentário: Dividindo a eluição em várias frações aumenta a eficiência de ligação / lavagens. <ol start="2"><li> Lava-se a resina 5 min com tampão de P30 (P5 tampão com uma concentração final de 30 mM de imidazol). </li><li> Repita o passo 6.2 2x e reunir as contas em um único tubo. Antes da última lavagem, remover tanto sobrenadante quanto possível. </li></ol> Comentário: Isto corresponde à amostra ligado GARRA (Figura 3B, pista 4). 7. A eluição da proteína purificada <ol><li> Incubar a resina TALON proteína ligada por 5 min sob rotação com P500 tampão (tampão P5 com uma concentração final de 500 mM de imidazol). Use uma proporção entre o buffer e gotas de 1:1 v / v (Por exemplo, se você tivesse tomado 200 mL de contas secas, você terá que adicionar 200 mL de P500). Repetir este passo duas vezes e manter cada fracção eluída separadamente (chamado de eluição 1 (E1), eluição 2 (E2) e eluição 3 (E3) e a figura 3B, pistas 5-7). </li></ol> Comentário: Os grânulos restantes podem ser utilizados para análise de eficiência de eluição (Figura 3B, pista 8). Tipicamente, 60-80% da proteína é eluída no total. <ol start="2"><li> Analisar a quantidade e qualidade da sua proteína purificada por carregar aproximadamente 20-40 ul de cada fracção com uma concentração padrão de BSA em SDS-PAGE e mancha com azul de Coomassie (Figura 3B) ou SYPRO mancha de proteína para visualização. </li></ol> Comentário: A proteína de interesse pode também ser detectado throughout o procedimento de purificação utilizando anticorpo anti-GST e anti-Seus anticorpos (figura 3C). 8. Armazenamento da Proteína Purificada <ol><li> Dializar a proteína purificada contra um tampão de armazenamento adequado (por exemplo, 20 mM de Tris-Acetato, pH 8,0, KAc 200 mM, Glicerol a 10%, EDTA 1 mM, DTT 0,5 mM) a 4 ° C. É importante verificar a precipitação de proteína purificada durante a diálise. Nós normalmente dialisar 2x durante 1 hora a 4 ° C, sob agitação de rotação. </li><li> Fazer pequenas aliquotas de proteína purificada (10-20 mL) e congelam-se em gelo seco durante 30 min. Armazenar as alíquotas a -80 ° C e evitar muitos ciclos de congelamento / descongelamento. </li></ol>

<ol>
	<li>Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J., Tan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+affinity+tags+for+protein+purification.">Comparison of affinity tags for protein purification.</a> Protein Expr. Purif. 41, 98-105 (2005).</li><li>Thain, A., Gaston, K., Jenkins, O., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+separation+of+GST+fusion+proteins+from+co-purifying+GroEL.">A method for the separation of GST fusion proteins from co-purifying GroEL.</a> Trends Genet. 12, 209-210 (1996).</li><li>Carr, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+recombinant+form+of+the+V+antigen+of+Yersinia+pestis,+using+three+different+expression+systems.">Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems.</a> Vaccine. 18, 153-159 (1999).</li><li>Armisen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adsorption+of+poly-His+tagged+glutaryl+acylase+on+tailor-made+metal+chelate+supports.">Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.</a> J. Chromatogr. A. 848, 61-70 (1999).</li><li>Kuhn, S., Zipfel, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculovirus+expression+vector+pBSV-8His+directs+secretion+of+histidine-tagged+proteins.">The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins.</a> Gene. 162, 225-229 (1995).</li><li>Buisson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+of+breast+cancer+proteins+PALB2+and+piccolo+BRCA2+in+stimulating+homologous+recombination.">Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.</a> Nat. Struct. Mol. Biol. 17, 1247-1254 (2010).</li><li>Filimonova, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoforms+of+Serratia+marcescens+nuclease.+The+role+of+Mg2++in+the+hydrolysis+mechanism.">Isoforms of Serratia marcescens nuclease. The role of Mg2+ in the hydrolysis mechanism.</a> Biochemistry. 62, 983-988 (1997).</li><li>Thorslund, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+breast+cancer+tumor+suppressor+BRCA2+promotes+the+specific+targeting+of+RAD51+to+single-stranded+DNA.">The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA.</a> Nat. Struct. Mol. Biol. 17, 1263-1265 (2010).</li><li>Genois, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+BRCA2+and+RAD51+for+promoting+homologous+recombination+in+Leishmania+infantum.">Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.</a> Nucleic Acids Res. 40, 6570-6584 (2012).</li><li>Shrestha, B., Smee, C., Gileadi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+vector+system:+an+emerging+host+for+high-throughput+eukaryotic+protein+expression.">Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression.</a> Methods Mol. Biol. 439, 269-289 (2008).</li></ol>

A GST-His protocolo de purificação aqui apresentada é adequada para a purificação de uma ampla gama de tamanhos de proteínas recombinantes: Foram purificados com sucesso Li RAD51 (41kDa), piBRCA2 (120kDa), PALB2 (130kDa), e um alto teor de proteína de peso molecular de instável Leishmania infantum: Li BRCA2 (125kDa) 6,9. Além disso, as proteínas purificadas com este protocolo foram bioquimicamente ativo 6. O sucesso deste método depende unicamente da expressão, solubil...

A purificação de proteínas recombinantes é crucial para tratar de questões fundamentais em bioquímica. As formas convencionais de purificação de proteínas, como a cromatografia de permuta iónica e cromatografia de exclusão de tamanho dependem de propriedades físicas da proteína-alvo, tais como o seu ponto isoeléctrico e carga ou tamanho, respectivamente. As características da proteína últimos são partilhados por uma variedade de proteínas, o que aumenta consideravelmente a probabilidade de as proteínas contaminantes, em estratégias de purificação de proteínas convencionais. Este problema pode ser contornado com o uso de várias colunas de purificação, o que é demorado. Ao mesmo tempo, os últimos métodos de cromatografia de exigir configuração experimental caro. A purificação por afinidade-tag aumenta fortemente a especificidade do alvo, tal como na maioria dos casos, a marcação será único para a proteína de interesse. Em estudos recentes, com Flag ou HA-purificação de afinidade tem sido amplamente usado. Em contraste com rec existenteprotocolos de purificação de proteínas ombinant em que tags simples são utilizados, foi estabelecida a combinação única de duas tags. O nosso método envolve a fusão de uma etiqueta de GST na extremidade N-terminal e uma cauda de His no terminal C da proteína de interesse, para uma óptima relação entre a quantidade e pureza da proteína desejada. A GST é uma marca longa (29 kDa), que é altamente eficiente para a purificação sobre Sepharose de glutationa. Além disso, utilizando GST garante boa relação custo-eficácia de nosso método 1. A possibilidade de se clivar a GST com o enzima PreScission (com a sequência de reconhecimento LeuGluValLeuPheGln / GlyPro, resultando na adição de apenas dois aminoácidos) tem muitas vantagens, por exemplo, esta estratégia evita a alteração das funções fisiológicas de proteínas devido a impedimento alostérico. O pequeno His-tag fundido com o outro extremo da proteína serve num segundo passo de purificação para aumentar a pureza da proteína por lavando a GST clivada, bem como proteínas degradadas e outros<html> contaminantes. Além disso, o protocolo não requer um passo de diálise quando se muda de GST para a coluna passo His-purificação (resina TALON). Contaminantes comuns em tais processos de purificação são proteínas de choque térmico (HSP). A adição de um passo de incubação com MgCl2 e ATP permite a remoção desses contaminantes (Figura 1). Fast Protein Liquid Chromatography (FPLC) e cromatografia líquida de alta performance (HPLC) são técnicas comuns que dependem instrumentação dispendiosa para gerar um rendimento elevado de proteína purificada de interesse. O protocolo de purificação de lote apresentamos aqui, em contraste, é manual e não necessita de uma instalação instrumental caro. Um rendimento elevado de proteína pode ser alcançado com a expansão do protocolo. Ao mesmo tempo, com o protocolo de purificação do lote, o volume de eluição pode ser ajustada, a fim de aumentar a concentração de proteína, o que não é dada com FPLC ou HPLC. ntent &quot;&gt; Além disso, com a GST-His protocolo de purificação do lote, proteínas com uma gama de dimensões de ~ 10-300 kDa pode ser purificada. Uma das maiores vantagens é dada pelo facto de que se pode purificar várias proteínas ao mesmo tempo , por exemplo, tanto um de tipo selvagem e a sua proteína mutante. O sucesso do protocolo apresentado apenas depende do nível de expressão e a solubilidade da proteína de interesse.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>E.Coli DH10Bac (competent)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus Expression System</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Maxi Prep Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Solution I and II</td>
		</tr>
		<tr>
			<td>Ultra Pure Bluo-Gal</td>
			<td>Gibco (Life)</td>
			<td>15519028</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gibco (Life)</td>
			<td>15529019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 infected cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>PreScission enzyme</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;s infected medium supplemented</td>
			<td>Gibco (Life)</td>
			<td>11605-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum characterized</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>P/S (Penicillin-Streptomycin)</td>
			<td>Gibco (Life)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose resin</td>
			<td>GE bioscience</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Talon resin</td>
			<td>Clontech</td>
			<td>635504</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>BioShop</td>
			<td>288324</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco (Life)</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal GST-antibody</td>
			<td>Production in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 6X-His-antibody</td>
			<td>Clontech</td>
			<td>631212</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dounce homogenizer (tight)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator (Fisher dismembrator)</td>
			<td>Fisher</td>
			<td>Model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (50 mm)</td>
			<td>Fisher Scientific</td>
			<td>2115217</td>
			<td></td>
		</tr>
	</tbody>
</table>

gst-his purification: a two-step affinity purification protocol yielding full-length purified proteins

Ensaios de chave em enzimologia para a caracterização bioquímica de proteínas in vitro, necessitam de elevadas concentrações de proteína purificada de interesse. Protocolos de purificação de proteína deve combinar eficiência, simplicidade e eficácia de custo 1. Aqui, nós descrevemos a GST-His método como um novo sistema de purificação por afinidade em pequena escala para as proteínas recombinantes, com base em um N-terminal de etiqueta de glutationa-Sepharose (GST) de 2,3 e uma marcação 10xHis C-terminal de 4, que são ambos fundida para a proteína de interesse. A última construção é usado para gerar baculovírus, para a infecção de células Sf9 infectadas para a expressão da proteína 5. GST é uma marca bastante longo (29 kDa), que serve para garantir a eficiência da purificação. No entanto, pode influenciar as propriedades fisiológicas da proteína. Por isso, é subsequentemente clivado da proteína usando a enzima PreScission 6. A fim de assegurar a máxima pureza e para remover a GST clivada, nósadicionou-se uma segunda etapa de purificação por afinidade com base na comparativamente pequena de His. Mais importante, a nossa técnica é baseada em duas etiquetas diferentes flanqueiam as duas extremidades da proteína, que é uma ferramenta eficaz para remover as proteínas degradadas e, portanto, enriquece proteínas de comprimento total. O método aqui apresentado não requer uma configuração instrumental caros, tais como FPLC. Além disso, incorporamos MgCl2 e lavagens de ATP para remover impurezas da proteína de choque térmico e tratamento nuclease abolir contaminando ácidos nucléicos. Em resumo, a combinação de duas marcas diferentes flanqueiam o N-e C-terminal e a capacidade para clivar um dos marcadores, garante a recuperação de uma proteína altamente purificada e de comprimento completo de interesse.

A fim de ilustrar a eficácia da GST-His protocolo de purificação, que purificado Rec14, um S. pombe proteína de 32,9 kDa. O Rec14 cDNA foi clonada no nosso vector pFastBac1 modificado permitindo a adição de GST-e Seus-tags no N-e C-terminais, respectivamente, (Figura 1A). Baculovírus recombinantes foram então preparados e utilizados para infectar células SF9 infectadas para a expressão da proteína. Os lisados ​​celulares solúveis foram incubados com contas de GST e proteínas li...

Watch this Scientific Journal Video about GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins at JoVE.com

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

No presente protocolo, demonstramos um método altamente eficiente e de baixo custo purificação de proteínas em pequena escala, que permite a purificação de proteínas recombinantes, combinando de forma única um GST-tag quebrável e uma pequena His-tag.

GST-Sua purificação: A Two-step Affinity Purification Protocolo Cedendo Full-length proteínas purificadas

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

gst-his-purification-two-step-affinity-purification-protocol-yielding

Research

JoVE Journal

Biology

41.2K visualizações.  Hôtel-Dieu de Québec.  No presente protocolo, demonstramos um método altamente eficiente e de baixo custo purificação de proteínas em pequena escala, que permite a purificação de proteínas recombinantes, combinando de forma única um GST-tag quebrável e uma pequena His-tag. 

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

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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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

Biologia

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.

Purificação da Hsp104, um Disaggregase Protein

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

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificação cromatográfica de Ribossomos Yeast Highly Active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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.

Purificação de Proteínas ortogonais Facilitado por um Tag Affinity Pequenas Bispecific

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 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 ...

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 ...

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 rendimento de purificação de afinidade com tag Proteínas 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 ...

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.

Método de purificação de proteína livre de Determinação Affinity Binding por termoforese Microscale

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

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

Isolamento de hepatócitos humanos por duas etapas Collagenase Perfusão Procedimento

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

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. ...