Este projeto foi financiado pelo Knut e Alice Wallenberg Foundation eo Conselho de Pesquisa sueco.

1. Clonagem da fusão alvo gene/ABDz1-tagged construir <ol><li> Prepare fragmentos de PCR do gene ABDz1 ladeado por sítios de restrição adequados para N-terminal ligadura do gene alvo na expressão plasmídeo (um plasmídeo contendo ABDz1 está disponível gratuitamente através de um acordo de transferência de material). </li><li> Cleave expressão purificada vector e fragmentos de PCR com enzimas de restrição escolhida em um buffer de reacção adequados. Purificar os produtos antes da ligadura. </li><li> Ligadura do fragmento ABDz1 restrito no vetor de expressão contendo o gene de interesse e transformar o produto ligadura para E. coli (no método original da cepa RR1ΔM15 foi usado 8). Spread células transformadas em placas de ágar suplementado com antibióticos adequados para a seleção. </li><li> PCR tela algumas colónias e seqüência verificar a cassete de expressão resultante por seqüenciamento de DNA. </li><li> Prepare plasmídeo de uma cultura overnight de um verif seqüênciacolônia de IED e transformar a cepa expressão preferida (no método original E. coli Rosetta (DE3), que hospeda um pRARE plasmídeo para melhorar a produção de proteínas codificadas por genes humanos, foram usados). </li></ol> 2. Expressão da proteína <ol><li> Inocular uma única colônia de bactérias em 10 ml de caldo tripticase soja suplementado com extrato de levedura 5 g / l, e os antibióticos apropriados. Incubar a 150 rpm a 37 ° C durante a noite. </li><li> Inocular 1 ml da cultura durante a noite em 100 ml de meio fresco e induzir a expressão da proteína (originalmente 1 mM isopropil-β-D-thiogalactoside quando um operon lac foi utilizado), quando as células atingem a fase de crescimento logarítmica. </li><li> Incubar a 150 rpm a 25 ° C durante a noite células e colheita por centrifugação. </li></ol> 3. Purificação de afinidade ortogonais <ol><li> Ressuspender o pellet em 25 ml de tampão em execução (25 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0,05% (w / v) Tween 20, pH 8,0) e interromper ªcélulas e por sonicação na amplitude de 60% e 1.0/1.0 pulsos por 3 min. Centrifugar a amostra e filtrar o sobrenadante contendo proteína-alvo (0,45 mm) antes de purificação suplementar. </li><li> Equilibrar um 1 ml HSA coluna Sepharose ou uma coluna de NHS-ativado, previamente combinada com HSA acordo com as recomendações do fornecedor, com 10 volumes de coluna (CV) de tampão de corrida em 1 ml / min em um sistema adequado de proteína de purificação. Carregue o lisado bacteriano de 0,5 ml / min e em seguida, redefinir a taxa de fluxo de 1 ml / min. </li><li> Lavar a coluna com 5 CV de tampão de corrida seguido por 5 CV de tampão de lavagem (5 mM NH 4 Ac, pH 5,5). </li><li> Eluir a amostra com tampão de eluição (0,5 M HAc, pH 2,5) a 1 ml / min e coletar frações, a absorção de monitor em 280 nm para selecionar frações do pico eluído para purificação adicional. </li><li> Piscina das frações com a maior concentração de proteína, dilua duas vezes em tampão de corrida Claro (20 de fosfato mM, 150 mM NaCl, pH 7,2) ecertifique-se o pH está em torno de neutro. Adicionar 1 M Tris-HCl pH 8 para aumentar o pH, se necessário. </li><li> Carga da amostra em 0,5 ml / min em uma coluna de um MabSelect HiTrap ml Claro que foi equilibrado, com 10 CV de tampão de corrida Claro em 1 ml / min. Redefinir a taxa de fluxo de 1 ml / min após o carregamento. </li><li> Lavar a coluna com 5 CV de tampão de corrida Claro (passo 5) e eluir as proteínas com 0,2 M HAc, pH 2.7. Para as proteínas-alvo sensíveis o eluído pode ser neutralizado diretamente no momento da recolha pela adição de Tris-HCl. </li></ol> Se as etapas cromatográficas são invertidos, o eluato da coluna MabSelect certeza pode ser diluído em tampão de corrida (passo 3.1) e do pH aumentou para cerca de 8 por adição de 1 M Tris-HCl. Em geral, os picos mais estreitos são observadas quando a matriz Claro é empregado na segunda etapa. 4. Avaliação de pureza por eletroforese de sódio dodecil sulfato de gel de poliacrilamida (SDS-PAGE) <ol><li> Carga das frações purificadas em umredução da SDS-PAGE e, se desejado, analisar o peso molecular do produto purificado por espectrometria de massa. É importante reduzir completamente a amostra uma vez que uma cisteína livre no ABDz1 pode causar dimerização do produto em condições não-redução. </li></ol> 5. Resultados representativos: Como uma prova de princípio, a purificação de afinidade ortogonais facilitada pela tag ABDz1 foi avaliada por três proteínas alvo humano que representam as classes de solubilidade diferentes, pesos moleculares e pontos isoelétricos (Tabela 1). O gene ABDz1 foi geneticamente fundido aos genes-alvo e as construções foram expressas em E. coli, Figura 2 mostra um fluxograma para todas as etapas no método consecutivamente. Após a purificação pelo protocolo ortogonais, amostras coletadas em diferentes pontos foram analisados ​​por SDS-PAGE (Figura 3). Os resultados mostram claramente a utilidade de uma tag dupla e duas etapas de purificação altamente específicos. Apesar de pureza razoável é achieved após a etapa de purificação inicial, visto a partir do gel, o passo sucessivo produz um produto muito puro bem adequado para aplicações altamente exigentes. <img alt="Figura 1" src="/files/ftp_upload/3370/3370fig1.jpg" /> Figura 1. Design da biblioteca combinatória usados ​​para a seleção do tag ABDz1 bispecific. Os 46 aminoácidos albumina de ligação de dobras de domínio em um pacote hélice estável três e ele contém um sítio de ligação para albumina sérica humana localizadas principalmente à hélice segundo. Por geneticamente randomizing eleven superfície exposta aminoácidos localizados na hélice de primeira e terceira, uma nova superfície de ligação foi projetado. Os onze posições são indicadas na figura e numerados de acordo com Kraulis et al. 9. Seguintes biopanning contra um dímero do Z-domínio da proteína A com a biblioteca combinatorial expressa em fago, a molécula ABDz1 bispecific foi identificado. <img alt = &quot;Figura 2&quot; src = &quot;files/ftp_upload/3370/3370fig2.jpg /&quot; /&gt; Figura 2. Um fluxograma simplificado para o protocolo ortogonais afinidade purificação. A PCR de fragmento contendo a seqüência ABDz1 é ligada (N-terminal), com um gene de interesse em um vetor de expressão adequado. O produto ligadura é transformado em E. coli para a verificação de seqüência e preparação plasmídeo. Após a transformação para um host de expressão, uma cultura em grande escala para a expressão da proteína recombinante é criado a partir de uma cultura inicial durante a noite. As bactérias são colhidas por centrifugação e lisadas por sonicação para produzir lisado bacteriano contendo a proteína alvo. Depois de uma etapa de filtração para remover partículas sólidas residuais, o lisado é submetido a purificação de afinidade ortogonal em um sistema de manipulação de líquidos submetendo-se a dois passos de purificação em sucessão. Picos eluídos de ambas as etapas de purificação são amostrados e avaliados por SDS-PAGE para avaliar a pureza ea comparação com tlisado ele antes de purificação. <img alt="Figura 3" src="/files/ftp_upload/3370/3370fig3.jpg" /> Figura 3. SDS-PAGE análise de proteínas-alvo expressa e purificada em fusão com ABDz1. Amostras retiradas do lisado bacteriano de três proteínas expressas em fusão para ABDz1, representando diferentes propriedades, ea tag ABDz1 si foram coletados junto com as amostras dos picos correspondentes da purificação em uma coluna HSA-seguido por um MabSelect Claro-coluna (A) . Lanes 1-4 representam amostras de lisados ​​antes de purificação, pistas 08/05 do HSA-purificação (primeira etapa) e 12/09 pistas da purificação MabSelect Claro (segunda etapa). Amostras são carregadas na seguinte ordem: ABDz1-141377, ABDz1-HT875, HT2375-ABDz1 e ABDz1. Além disso, amostras adquiridas a partir da mesma lisados ​​purificados na ordem inversa nas mesmas colunas foram analisados ​​(B). Lanes 1-3 representam amostras de lisados ​​antes de purificação, faixas 4-6 from MabSelect purificação-Sure (primeira etapa) e 09/07 a partir de pistas HSA-purificação (segunda etapa). As amostras foram carregados na mesma ordem como em (A), mas a marca em si não está incluída. A partir desses resultados, é claro que este método de duas etapas produz proteínas altamente purificadas, independentemente da ordem em que as etapas são aplicadas. <table border="1"><tbody><tr><td> B nome </td><td> Proteína alvo UniProt c </td><td> Molecular peso (kDa) </td><td> Solubilidade classe d </td><td> Peso molecular de produto de fusão (kDa) </td><td> Ponto isoelétrico de produto de fusão </td></tr><tr><td> 141377 </td><td> B7Z315 </td><td> 17,4 </td><td> 4 </td><td> 23,7 </td><td> 8,5 </td></tr><tr><td> HT875 </td><td> P01040 </td><td> 10,8 </td><td> 3 </td><td> 17,1 </td><td> 4,9 </td></tr><tr><td> HT2375 </td><td> P00740 </td><td> 8,9 </td><td> 5 </td><td> 15,2 </td><td> 6,8 </td></tr></tbody></table><ol class="lalpha"><li> Peso molecular de ABDz1 tag sozinhos é de 6,3 kDa, ponto isoelétrico 6,7. </li><li> Proteína-alvo representa uma parte da proteína UniProt. </li><li> <a href="http://www.uniprot.org">http://www.uniprot.org</a> . </li><li> Classe de solubilidade de um fragmento de proteína com N-terminal Seu seis ABP 10. </li></ol> Tabela 1. Proteínas-alvo e produtos de fusão com uma tag ABDz1 avaliada em um estudo de prova de princípio. Três únicas proteínas humanas com peso molecular diferente, solubilidade e ponto isoelétrico foram escolhidos para a expressão e purificação pela abordagem afinidade ortogonais.

<ol>
	<li>Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+most+of+affinity+tags.">Making the most of affinity tags.</a> Trends Biotechnol. 23, 316-320 (2005).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+strategies+for+selective+protein+purification.">Protein engineering strategies for selective protein purification.</a> CET. 28, 1315-1325 (2005).</li><li>Porath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+chelate+affinity+chromatography,+a+new+approach+to+protein+fractionation.">Metal chelate affinity chromatography, a new approach to protein fractionation.</a> Nature. 258, 206-213 (1975).</li><li>Linhult, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+the+interaction+between+albumin-binding+domain+from+streptococcal+protein+G+and+human+serum+albumin.">Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin.</a> Protein Sci. 11, 206-213 (2002).</li><li>Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+IgG-binding+domain+based+on+staphylococcal+protein.">A synthetic IgG-binding domain based on staphylococcal protein.</a> A. Protein Eng. 1, 107-113 (1987).</li><li>Alm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+bispecific+protein+selected+for+orthogonal+affinity+purification.">A small bispecific protein selected for orthogonal affinity purification.</a> Biotechnol. J. 5, 605-617 (2010).</li><li>Nilsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+affinity+domains+for+the+detection,+purification+and+immobilization+of+recombinant+proteins.">Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.</a> J. Mol. Recognit. 9, 585-594 (1996).</li><li>R&#252;ther, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pUR+250+allows+rapid+chemical+sequencing+of+both+DNA+strands+of+its+inserts.">pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.</a> Nucleic Acids Res. 10, 5765-5772 (1982).</li><li>Kraulis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serum+albumin-binding+domain+of+streptococcal+protein+G+is+a+three-helical+bundle:+a+heteronuclear+NMR+study.">The serum albumin-binding domain of streptococcal protein G is a three-helical bundle: a heteronuclear NMR study.</a> FEBS Lett. 378, 190-194 (1996).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+flow+cytometry-based+method+for+analysis+of+expression+levels+in+E+coli+giving+information+about+precipitated+and+soluble+protein.">Novel flow cytometry-based method for analysis of expression levels in E coli giving information about precipitated and soluble protein.</a> J. Biotechnol. 119, 133-146 (2005).</li><li>Carter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+proteolysis+of+fusion+proteins.">Site-specific proteolysis of fusion proteins.</a> ACS Sympos. Ser. 427, 181-193 (1990).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively+charged+purification+tags+for+selective+anion-exchange+recovery.">Negatively charged purification tags for selective anion-exchange recovery.</a> PEDS. 17, 779-786 (2004).</li><li>Gr&#228;slund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+engineering+of+a+protein+domain+to+allow+efficient+ion-exchange+recovery.">Charge engineering of a protein domain to allow efficient ion-exchange recovery.</a> Protein Eng. 13, 703-709 (2000).</li></ol>

Não há conflitos de interesse declarados.

O protocolo de purificação de ortogonais afinidade apresentada aqui permite purificação eficiente de uma ampla gama de proteínas-alvo. Ao combinar o local de ligação inerente do domínio albumina de ligação com uma nova superfície de ligação, uma pequena proteína tag bispecific foi desenvolvido. É muito simples de utilizar a tag uma vez que pode simplesmente ser clonados e expressos em fusão a qualquer proteína de interesse em qualquer vetor de expressão preferida. Equipamento padrão disponível na maioria dos laboratórios pode ser empregado para a purificação de proteínas em duas etapas. Uma vez que a função da tag ABDz1 depende de dobramento correto do domínio para expor sua eficiência duas superfícies de ligação, o método é limitado a proteínas expressas na forma solúvel e não adequado para purificações em condições de desnaturação. Agentes redutores também devem ser evitados, uma vez que interfere com a ligação de ABDz1 para o Z-domínio sobre a matriz MabSelect Claro. Ortogonais afinidade purificaçãoção fornece uma alternativa útil aos métodos tradicionais, a fim de satisfazer as exigências de proteínas altamente purificadas em muitas aplicações exigentes. Ao mesmo tempo, este método elimina a necessidade de marcação combinatória quando as estratégias de purificação diferentes são usados ​​em sucessão. Para aplicações que requerem uma proteína alvo nativo, um sítio de clivagem da protease podem ser introduzidas para tornar a remoção enzimática da tag ABDz1 possível 11. Além disso, a tag ABDz1 é o domínio primeira proteína bispecific pequenas e dobradas descritas até o momento. Ele é único, uma vez que fornece uma estratégia de purificação que depende de duas alvo interações afinidade específica. No futuro seria interessante para expandir este conceito através do desenvolvimento de novas etiquetas bispecific que carregam novas superfícies de ligação que são compatíveis com prontamente disponíveis e baratos resinas cromatográficas. Por exemplo, substituindo os aminoácidos envolvidos na ligação da albumina com resíduos básicos, uma tag compatível com iem cromatografia de troca pode ser obtida. Um conceito similar tem se mostrado eficaz pela engenharia responsável pela Z-domínio para gerar etiquetas conhecido como ácido Z 12 e 13 básicas Z compatível com anion e troca catiônica, respectivamente.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> E. coli RR1ΔM15 </td><td> Americana Culture Collection Tipo </td><td> 35102 </td></tr><tr><td> E. coli Rosetta (DE3) </td><td> Novagen </td><td> 70954 </td></tr><tr><td> Caldo tripticase soja </td><td> Difco </td><td> 211822 </td></tr><tr><td> Extrato de levedura </td><td> Difco </td><td> 212720 </td></tr><tr><td> Vibra sonicador celular </td><td> Sonics e materiais </td><td> - </td></tr><tr><td> HSA Sepharose </td><td> Pharmacia Biotech </td><td> Produto ex- </td></tr><tr><td> HiTrap MabSelect Claro </td><td> GE Healthcare </td><td> 11-0034-93 </td></tr><tr><td> HiTrap NHS-activated coluna HP </td><td> GE Healthcare </td><td> 17-0716-01 </td></tr></tbody></table>

orthogonal protein purification facilitated by a small bispecific affinity tag

Devido aos elevados custos associados com a purificação de proteínas recombinantes os protocolos precisam ser racionalizadas. Para high-throughput esforços há uma demanda por métodos gerais que não necessitam de proteína-alvo específicos de otimização 1. Para alcançar este objectivo, tags purificação que geneticamente pode ser fundida ao gene de interesse são comumente usados ​​2. A alça de afinidade mais utilizado é o tag hexa-histidina, que é adequado para a purificação sob as duas condições nativas e desnaturantes 3. A carga metabólica para a produção da marca é baixo, mas ele não fornece a alta especificidade como concorrentes afinidade 1,2 estratégias de cromatografia base. Aqui, uma tag de purificação bispecific com dois diferentes sítios de ligação em um ácido amino 46, de domínio pequena proteína tem sido desenvolvido. O domínio de ligação de albumina é derivada de proteína G estreptocócicas e tem uma forte afinidade inerente à albumina sérica humana(HSA). Onze superfície exposta aminoácidos, que não estão envolvidos em albumina de ligação de 4, foram randomizados geneticamente para produzir uma biblioteca combinatória. A biblioteca de proteína com o romance dispostas aleatoriamente superfície de ligação (Figura 1) foi expressa em partículas phage para facilitar a seleção de pastas pelo fago tecnologia de exibição. Através de várias rodadas de biopanning contra um dimérica Z domínio derivado da proteína estafilocócica A 5, uma molécula pequena, bispecific com afinidade para ambos HSA eo novo alvo foi identificado 6. O domínio de nova proteína, denominada ABDz1, foi avaliada como uma marca de purificação para uma seleção de proteínas-alvo com peso molecular diferente, solubilidade e ponto isoelétrico. Três proteínas-alvo foram expressas em Escherichia Escherishia com a tag romance fundidos aos seus N-terminais e, posteriormente, purificadas por afinidade. Purificação inicial em uma coluna com HSA imobilizados ou Z-domínio resultou em relatiprodutos vely puro. Duas etapas de purificação de afinidade com a tag bispecific resultaram em melhoria substancial da pureza da proteína. Media cromatográfica com a Z-domínio imobilizada, por exemplo MabSelect Claro, estão prontamente disponíveis para a purificação de anticorpos e HSA pode facilmente ser quimicamente ligado a meios de comunicação para fornecer a segunda matriz. Este método é especialmente vantajosa quando há uma alta demanda sobre a pureza da proteína-alvo recuperado. O bifunctionality da tag permite que dois diferentes passos cromatográficos para ser usado enquanto a carga metabólica no host expressão é limitada devido ao pequeno tamanho da tag. Ele fornece uma alternativa competitiva aos chamados tagging combinatória, onde várias marcas são usados ​​em combinação 1,7.

Watch this Scientific Journal Video about Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag at JoVE.com

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Um romance e altamente eficiente em duas etapas cromatografia de afinidade protocolo foi desenvolvido e é descrita em detalhes. O método é baseado em uma tag de purificação com duas pequenas afinidades inerentes e é aplicável a uma grande variedade de proteínas-alvo com propriedades diferentes.

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 ...

orthogonal-protein-purification-facilitated-small-bispecific-affinity

Research

JoVE Journal

Biology

16.9K visualizações.  Royal Institute of Technology. Um romance e altamente eficiente em duas etapas cromatografia de afinidade protocolo foi desenvolvido e é descrita em detalhes. O método é baseado em uma tag de purificação com duas pequenas afinidades inerentes e é aplicável a uma grande variedade de proteínas-alvo com propriedades diferentes.

Vídeo: Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

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

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolamento de proteínas lábeis Multi-Complexos por In vivo Controlled Cellular Cross-Linking and Imuno-magnética cromatografia de afinidade

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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 ...

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 ...

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

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

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

GST-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. ...

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 ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Um protocolo para exibição Fago e Affinity Seleção Usando Iscas proteína recombinante

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

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. ...