Este trabalho foi financiado por uma concessão do NIH (5T32GM008275-22) e um American Heart Association predoctoral comunhão (para EAS); uma bolsa de interface Química Biologia a partir do NIH (2T32GM071339-06A1) (para MED); e subvenções do NIH (1DP2OD002177-01 e NS067354-0110), The Ellison Medical Foundation e The Bill and Melinda Gates Foundation (a JS).

1. Expressão de Hsp104 <ol><li> O plasmídeo utilizado para a purificação em E. coli, pPROEX-HTB-Hsp104, contém o Hsp104 estrutura aberta de leitura sob o controle do promotor induzível trc 26. O plasmídeo produz Hsp104 com um N-terminal Seu 6 tag que pode ser removido por clivagem da protease TEV. PPROEX transformar-HTB-Hsp104 em códon otimizado E. coli BL21-CodonPlus-RIL células (Stratagene, Agilent Technologies) usando um procedimento típico de transformação bacteriana (por exemplo, de acordo com as instruções do fabricante). É importante usar um E. códon otimizado coli cepa Hsp104 porque tem um viés códon incomum. </li><li> Inocular uma cultura 100mL 2XYT (USB. Receita: 16g / L de caseína peptona, 10g / L de extrato de levedura, 5g / L NaCl, pH 7,0) suplementado com ampicilina e cloranfenicol 100μg/mL 34μg/mL com transformantes fresco e crescer durante a noite a 37 ° C com agitação de 200 rpm. </li><li> Inocular 10 ml da cultura durante a noite em 6 X 1L 2XYT com 100μg/mL ampicilina e cloranfenicol 34μg/mL em frascos de 2L. Agite a 250rpm a 37 ° C até OD 600 = ,4-0,6. Parar de tremer e reduzir a temperatura para 15 ° C. Permitem que as células para equilibrar sem agitação por 30min na incubadora até atingir 15 ° C. Uma vez que 15 ° C é atingido induzir a expressão da proteína pela adição de IPTG a uma concentração final de 1mM. Retomar a agitação a 250rpm durante a noite (12-16 horas). </li></ol> 2. Colheita de células e de Lise <ol><li> Células colheita por centrifugação (em um rotor pré-resfriada) a 4.000 rpm por 20min a 4 ° C (usamos um Sorvall RC 3BP centrífuga +). Etapas subseqüentes devem ser realizados imediatamente, porque Hsp104 atividade é diminuída quando E. células coli são congelados. Além disso, todas as etapas subseqüentes devem ser realizados no gelo ou a 4 ° C. </li><li> Ressuspender pellets de células em prechilled (no gelo) 10 ml tampão de lise (40mm HEPES-KOH pH 7,4, 500mM KCl, 20mM MgCl 2, 2,5% (w / v) de glicerol, 20mM imidazol, 5m pepstatin A, cocktail inibidor da protease completa (1 EDTA sem tablet/50mL), e 2mM β-mercaptoetanol). </li><li> Lisar as células com uma imprensa francesa homogeneizador (Emulsiflex) que usa um trocador de calor imerso em água gelada. Antes da utilização, garantir que todos os aglomerados celulares foram solubilizados. Depois de equilibrar homogeneizador com tampão de lise, duas passagens do cilindro com uma pressão de 15,000-18,000 psi é suficiente para a lise completa. Se uma imprensa francesa não está disponível, a incubação com a lisozima seguido de sonicação pode ser usado (ver discussão). Salvar uma pequena amostra de células lisadas por SDS-PAGE análise (1μL) (Lysate na figura. 2). </li><li> Remover restos celulares por centrifugação a 16.000 rpm por 20min a 4 ° C (usamos um RC5C Sorvall centifuge +). Reter sobrenadante para o próximo passo e salvar uma pequena amostra (1μL) do sobrenadante para análise SDS-PAGE (Ni Load na figura. 2). </li></ol> 3. Purificação Hsp104 <ol><li> Mix sobrenadante do passo 2.4 com 12 ml (2 ml Ni-Sepharose pérolas por 1L de células) de dejetos de 50% de tampão de lise equilibrada Ni Sepharose-beads (GE). </li><li> Rode a amostra lentamente por 3 horas a 4 ° C tal que Ni-Sepharose permanece uniformemente suspensa e espuma é minimizado. Menor tempo de incubação são possíveis, mas vai diminuir o rendimento. A 4 ° C na presença de inibidores de protease, a degradação ocorre pouco, e um tempo de incubação 3 horas Ni Sepharose-não diminui a atividade do produto final. Coletar Ni-Sepharose por centrifugação por 2 min a 4 ° C a 2.000 rpm (Eppendorf 5810R centrífuga). Salvar uma pequena amostra do sobrenadante para análise SDS-PAGE (1μL) e descartar o resto (Ni Flow Through (FT) na figura. 2). </li><li> Lavar os recuperados Ni Sepharose com 25 volumes de coluna de tampão de lavagem (40mm HEPES-KOH pH 7.4, 150mm KCl, 20mM MgCl 2, 2,5% (w / v) de glicerol, 20mM imidazol, 2mM β-mercaptoetanol), 5 volumes da coluna tampão de lavagem com KCl 1M para remover os contaminantes ligados eletrostaticamente para Hsp104, e 25 volumes de coluna de tampão de lavagem. Coletar Ni-Sepharose após cada lavagem por centrifugação por 2 min a 4 ° C a 2.000 rpm (Eppendorf 5810R centrífuga). Após cada ciclo de lavagem e centrifugação, remover tampão por aspiração. </li><li> Para eluir Hsp104, misture Ni-Sepharose com 1mL tampão de eluição (40mm HEPES-KOH pH 7.4, 150mm KCl, 20mM MgCl 2, 2,5% (w / v) de glicerol, imidazol 350mm, 2mM β-mercaptoetanol) por 1 ml Ni-Sepharose e girar a 4 ° C por 20 minutos. A concentração de imidazol alta interrompe o talão Ni-6 Sua interação. Remover Ni-Sepharose com colunas vazias 1,2 mL de rotação (Bio-Rad) por centrifugação por 2 min a 4 ° C a 2.000 rpm (Eppendorf 5810R centrífuga). Salvar 1μL de eluato para análise SDS-PAGE (Ni eluato na figura. 2). Para cada litro de células, ~ 15mg de Hsp104 é obtido nesta etapa. </li><li> Tampão eluído em troca de buffer Q (20mM Tris-HCl pH 8, EDTA 0,5 mM, MgCl 5mM 2, 50 mM NaCl), utilizando MWCO 30.000 Amicon Ultra (Millipore) 15mL unidades concentrador centrífugo. Primeiro, concentrado protéico de ~ 1,5 ml em seguida, adicione 14.5mL de tampão para diluir proteína Q 10 vezes. Repita 3 vezes para a troca de buffer. O pH aumentou e baixo teor de sal garante que Hsp104 tem uma alta carga negativa. </li><li> Purify Hsp104 através de troca aniônica cromatografia usando um tampão Q equilibrada Resource Q coluna 6ml (GE). Antes da injeção, a proteína é filtrada através de uma baixa ligação protéica Millex GP PES Membrana 0.22μM seringa filtro (Millipore). Nós empregamos uma vazão de 1mL/min. Lavar fracamente proteínas de ligação com 1 volume de tampão de coluna 20% Q + (20mM Tris-HCl pH 8, EDTA 0,5 mM, MgCl 2 5mM, 1M NaCl). Eluir Hsp104 com gradiente linear (20% -50% de buffer Q +) mais de 5 volumes da coluna (Fig. 3). Coletar frações de 1ml. Hsp104 e mais variantes tipicamente eluída no NaCl ~ 400mm (31mS/cm). Salvar amostras de frações de pico (1μL) para análise de SDS-PAGE (Fig. 3, inset). </li><li> Para remover sua 6 tag, proteína de troca utilizando o concentrador Amicon centrífuga como descrito acima (veja o passo 3.5) em tampão de clivagem (20mM HEPES-KOH pH 7.4, 140mm KCl, 10mM MgCl e 2). Use proTEV protease (Promega) ou AcTEV protease (Invitrogen) de acordo com instruções do fabricante. Para Hsp104, descobrimos que os rácios mais elevados de protease TEV: Hsp104 são necessários para a clivagem completa. Nós usamos uma proporção de 1 unidade de protease por 12μg Hsp104. Clivagem deve ser realizada a 30 ° C por 2-4 horas, seguido de incubação de 16 horas a 4 ° C. Hsp104 concentração final deve ser entre 20-75μM monômero. Destroem qualquer Hsp104 restantes His6-marcadas e protease TEV com Ni-Sepharose (GE), adicionando-Ni Sepharose em excesso para a quantidade de Hsp104 na reação de clivagem (assumir contas pode ligar 15mg de proteína por mL de resina embalado). Remover contas com colunas de spin vazia (Bio-Rad) por centrifugação de 2min a 2.000 rpm a 4 ° C. Coletar amostras antes e depois da clivagem (1μL) para SDS-PAGE análise para garantir clivagem, ea remoção de proteínas uncleaved (Fig. 4). </li><li> Em troca de tamanho do buffer de exclusão (20mM HEPES-KOH pH 7.4, 140mm KCl, 10mM MgCl 2, 0,1 mM EDTA, 1mM DTT) usando MWCO 30.000 Amicon Ultra (Millipore), como descrito no passo 3.5. </li><li> Purificar Hsp104 via tamanho-exclusão cromatografia. Use o tamanho do buffer Superose exclusão equilibrado 6 ou Superdex 200 coluna (ambos da GE) (Fig. 5). Antes da injeção, a proteína é filtrada através de uma baixa ligação protéica Millex GP PES Membrana 0.22μM seringa filtro (Millipore). Por menos de 10 mg de proteína, o 10/300 colunas tamanho (24mL) pode ser usado e frações de 0,5 ml são coletados. Para as amostras superiores a 10 mg, a coluna tamanho 12/60 (120ml) deve ser usado e 1 ml frações são coletadas. Consulte as instruções do fabricante para caudais e volumes de amostra a ser carregado. Após purificação, as frações Hsp104 são agrupados conforme mostrado na figura. 5 e concentrada em Unidades Concentrador Amicon centrífugas (Millipore). Hsp104 é armazenado como descrito abaixo. Devido à perda de material em separar Hsp104 produtos de degradação e contaminantes no rendimento final da purificação full-length Hsp104 é ~ 1-3mg por litro de partida cultura. </li></ol> 4. Hsp104 Atividade Disaggregase <ol><li> Após purificação, é uma boa prática para avaliar Hsp104 atividade em um ensaio de desagregação. Normalmente, nós empregamos um ensaio de reativação luciferase 2 (Fig. 6). Neste ensaio, Hsp104 em conjunto com o sistema chaperone Hsp70 (Hsp70 e Hsp40) desagrega-uréia desnaturado agregados luciferase firefly 2. Solúveis luciferase catalisa a oxidação da luciferina para oxiluciferina, uma reação que libera luz. Por luminescência de monitoramento, a reativação relativa, e, portanto, desagregação, da luciferase pode ser determinada. Hsp104 deve ser capaz de sinergicamente colaborar com o sistema de co-chaperone Hsp70. A quantidade de luminescência recuperado depende da Hsp70 exata: par Hsp40 utilizado. Empregamos rotineiramente Hsp72 e Hsp40 (Designs Assay). No mínimo, ativos Hsp104 deve produzir um aumento de 5 vezes na reativação da luciferase, quando comparado à reativação de Hsp72: Hsp40 sozinho (Fig. 6). Hsp104 preparações que não conseguem atingir esse nível de atividade são descartados. </li></ol> 5. Hsp104 Armazenamento <ol><li> De curto prazo de armazenamento, Hsp104 pode ser mantido a 4 ° C em tamanho-exclusão buffer. No entanto, a atividade vai diminuir depois de 2-3 dias a 4 ° C e acentuadamente após uma semana a 4 ° C. Para ensaios de desagregação recomendamos que Hsp104 deve ser usado o mais rapidamente possível após a purificação. Idealmente, Hsp104 deve ser utilizado imediatamente para ensaios de desagregação amilóide. </li><li> Se a proteína deve ser armazenado a longo prazo, Hsp104 é trocada dentro do buffer de armazenamento (20mM HEPES-KOH pH 7.4, 140mm KCl, 10mM MgCl 2, 0,1 mM EDTA, 1mM DTT, glicerol 10% (w / v)). Alíquotas são 100μl pressão congelados em nitrogênio líquido e armazenadas a -80 ° C. Ciclos de congelamento e descongelamento reduzir drasticamente Hsp104 atividadee deve ser evitado. Recomendamos lentamente descongelamento Hsp104 no gelo. Amilóide disaggregase-atividade irá diminuir drasticamente depois de 1 mês a -80 ° C. </li></ol> 6. Resultados representante e números: <img src="/files/ftp_upload/3190/3190fig1.jpg" alt="Figura 1"/> Figura 1. Hsp104 é um disaggregase bifuncional. Desagregação dos agregados desordenado (mostrado à esquerda) requer a cooperação do sistema chaperone Hsp70 (Hsp70 e Hsp40) 2. Remodela Hsp104 ordenou agregados amilóides (mostrado à direita) sem o auxílio de Hsp70 e Hsp40 in vitro, mas Hsp70 e Hsp40 pode melhorar Hsp104 atividade contra amilóide 26,28. Para ambos os tipos de estruturas de agregados, Hsp104 casais hidrólise de ATP a translocação do substrato através de seu canal central para promover a desagregação. Loops tirosina-bearing poros envolver e substrato de transporte através do canal central 49-52. <img src="/files/ftp_upload/3190/3190fig2.jpg" alt="Figura 2"/> Figura 2. SDS-PAGE análise de Ni-Sepharose etapa de purificação de afinidade. Lysate, Load Ni, Ni FT e amostras de eluato Ni foram fracionadas por SDS-PAGE usando um 40-20% Tris-HCl 1,0 milímetros Critério de gel (Bio-Rad) e corados Coomassie . Note que nem todos os Hsp104 é capaz de se ligar ao Ni-Sepharose. Ampla Faixa de marcadores de peso molecular (Bio-Rad) são mostrados (esquerda lane). <img src="/files/ftp_upload/3190/3190fig3.jpg" alt="Figura 3"/> Figura 3. Recurso de purificação Q de Ni-Sepharose eluído. Traço azul representa a absorvância a 280nm traço eo verde representa% buffer Q + (máximo de 50%). O primeiro pico, que é eluído em 20% Q + contém impurezas, produtos de degradação e Hsp104 indevidamente dobrado. O segundo pico e os principais contém Hsp104 correctamente dobrados e ativo. Taxa de fluxo foi 1mL/min. Gradiente de 20-50% Q + é de 30 minutos ou 5 volumes da coluna. Detalhe: frações de pico são resolvidos por SDS-PAGE análise usando um 40-20% Tris-HCl 1,0 milímetros Critério de gel (Bio-Rad) e corados Coomassie. Ampla Faixa de marcadores de peso molecular (Bio-Rad) são mostradas (esquerda). <img src="/files/ftp_upload/3190/3190fig4.jpg" alt="Figura 4"/> Figura 4. SDS-PAGE análise de proTEV etapa de clivagem da protease. Seus 6-Hsp104 de purificação Resource Q foi tratado com proTEV protease durante 4 horas a 30 ° C e 16 horas a 4 ° C. Amostras foram fracionadas por SDS-PAGE usando um 40-20% Tris-HCl 1,0 milímetros Critério de gel (Bio-Rad) e corados Coomassie. Note-se que proTEV clivada Hsp104 migra mais rapidamente. TEV protease e Hsp104 uncleaved foram esgotados com Ni-Sepharose, conforme descrito no Passo 3.7. Ampla Faixa de marcadores de peso molecular (Bio-Rad) são mostradas (esquerda). <img src="/files/ftp_upload/3190/3190fig5.jpg" alt="Figura 5"/> Figura 5. Tamanho-exclusão purificação de Hsp104. Clivadas Hsp104 foi ainda purificada por meio de uma coluna de filtração Superose 6 gel (10/300, GE). = Vazão 0.4ml/min. O pico entre as linhas tracejadas representam frações agrupadas. Detalhe: frações de pico são resolvidos por SDS-PAGE análise usando um 40-20% Tris-HCl 1,0 milímetros Critério de gel (Bio-Rad) e corados Coomassie. Ampla Faixa de marcadores de peso molecular (Bio-Rad) são mostradas (esquerda). <img src="/files/ftp_upload/3190/3190fig6.jpg" alt="Figura 6"/> Figura 6. Ensaio de reativação luciferase. Agregados desnaturadas firefly luciferase (50nm) foram incubadas com um Hsp72 e Hsp40 (1 Hm) (ambos dos projetos Assay), Hsp104 (6μM monômero) ou Hsp104, Hsp72 e Hsp40 de 90min a 25 ° C. Luciferase desnaturado só é totalmente reativado na presença de ambos os Hsp104 e Hsp40/Hsp72. Luminescência recuperado é medida em uma Infinito M1000 Leitor de placa (Tecan). Valores representam médias ± EPM (n = 3).

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Cronograma: Para a atividade Hsp104 máxima recomendamos que o sistema de purificação de todo ser concluída o mais rapidamente possível. No entanto, o número de etapas de purificação torna um calendário exigente, que nem sempre pode ser prático. Se as etapas de purificação são realizadas o mais rapidamente possível, o tempo a partir do final de expressão durante a noite até a 2-4 horas de incubação a 30 ° C com TEV protease é de aproximadamente 9-11 horas. Um lugar potencial para fazer uma paus...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> BL21-CodonPlus-RIL células competentes </td><td> Stratagene, Agilent Technologies </td><td> 230255 </td></tr><tr><td> 2XYT caldo </td><td> USB </td><td> 75864 </td></tr><tr><td> Completo, mini, EDTA livre inibidor da protease comprimidos </td><td> Roche </td><td> 1836170 </td></tr><tr><td> A Pepstatin </td><td> Sigma </td><td> P4265 </td></tr><tr><td> Ni-Sepharose 6 Fluxo Rápido </td><td> GE Healthcare </td><td> 17-5318-02 </td></tr><tr><td> Amicon Ultra-15 centrífuga unidades de filtro (MWCO 30.000) </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> Resource Q - coluna 6ml </td><td> GE Healthcare </td><td> 17-1179-01 </td></tr><tr><td> proTEV Protease </td><td> Promega </td><td> V6052 </td></tr><tr><td> AcTEV Protease </td><td> Invitrogen </td><td> 12575015 </td></tr><tr><td> Superose 6 10/300 GL </td><td> GE Healthcare </td><td> 17-5172-01 </td></tr><tr><td> Hsp40 </td><td> Designs ensaio </td><td> SPP-400 </td></tr><tr><td> Hsp72 </td><td> Designs ensaio </td><td> ADI-NSP-555 </td></tr></tbody></table>

purification of hsp104, a protein disaggregase

Hsp104 é um hexamérica AAA + 1 proteína de levedura, que acopla a hidrólise de ATP para a proteína desagregação 10/02 (Fig. 1). Esta atividade dá dois principais vantagens seletivas. Primeiro, renaturalização de agregados desordenada por Hsp104 capacita sobrevivência levedura após várias proteínas-misfolding salienta, incluindo choque térmico 3,5,11,12. Em segundo lugar, a remodelação das fibrilas de cross-beta-amilóide por Hsp104 permite levedura para explorar prions miríade (amilóides infecciosas) como um reservatório de benéfico e hereditárias variação fenotípica 13-22. Notavelmente, Hsp104 diretamente remodela oligômeros preamyloid e fibrilas amilóides, incluindo aqueles composto de proteínas de levedura prion Sup35 e Ure2 23-30. Esta funcionalidade amilóide remodelação é uma faceta especializados de levedura Hsp104. O E. orthologue coli, ClpB, não oligômeros remodelar preamyloid ou fibrilas amilóides 26,31,32. Orthologues Hsp104 são encontrados em todos os reinos da vida, exceto, perplexidade, os animais. De fato, se as células animais possuem qualquer sistema enzimático que os casais desagregação proteína para renaturalização (ao invés de degradação) permanece desconhecido 33-35. Assim, nós e outros propuseram que Hsp104 pode ser desenvolvido como um agente terapêutico para várias doenças neurodegenerativas relacionadas com o misfolding de proteínas específicas em oligômeros preamyloid tóxicos e fibrilas amilóides 4,7,23,36-38. Não existem tratamentos que visam directamente as espécies agregados associados a estas doenças. No entanto, se dissolve Hsp104 oligômeros tóxicos e fibrilas amilóides composto de alfa-sinucleína, que são conectados com Doença de Parkinson 23, bem como formas de amilóide de PrP 39. Importante, Hsp104 reduz a agregação da proteína e melhora a neurodegeneração em modelos de roedores da doença de Parkinson 23 e doença de Huntington 38. Idealmente, para otimizar o tratamento e minimizar os efeitos colaterais, Hsp104 seria projetado e potencializadas para seletivamente remodelar agregados específicos central para a doença em questão 4,7. No entanto, a limitada compreensão estrutural e mecânica de como Hsp104 desagrega um repertório tão diversificado de estruturas agregadas e proteínas não relacionadas frustra estes esforços 30,40-42. Para compreender a estrutura e mecanismo de Hsp104, é essencial para estudar a proteína pura e reconstituir a sua actividade disaggregase com componentes mínimos. Hsp104 é uma proteína 102kDa com um pI de aproximadamente 5,3, que hexamerizes na presença de ADP ou ATP, ou em concentrações da proteína na ausência de 43-46 nucleotídeos. Aqui, nós descrevemos um protocolo otimizado para a purificação de altamente ativa, Hsp104 estável de E. coli. O uso de E. coli simplificado permite produção em larga escala e nosso método pode ser realizado de forma rápida e confiável para inúmeras Hsp104 variantes. Nosso protocolo aumenta Hsp104 pureza e simplifica a sua remoção 6 tag em comparação com um método de purificação prévia do E. coli 47. Além disso, o nosso protocolo é mais fácil e conveniente do que dois protocolos mais recentes 26,48.

Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

Purification of Hsp104, a Protein Disaggregase

Aqui, nós descrevemos um protocolo para a purificação de altamente ativa Hsp104, a AAA hexamérica + proteína de levedura, que acopla a hidrólise de ATP a desagregação de proteína. Este esquema explora uma construção His6 marcadas para a purificação de afinidade<em&gt; E. coli</em&gt; Seguido de cromatografia de troca aniônica, His6 tag-remoção com protease TEV e tamanho-exclusão cromatografia.

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 ...

purification-of-hsp104-a-protein-disaggregase

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Biology

17.3K visualizações.  University of Pennsylvania. Aqui, nós descrevemos um protocolo para a purificação de altamente ativa Hsp104, a AAA hexamérica + proteína de levedura, que acopla a hidrólise de ATP a desagregação de proteína. Este esquema explora uma construção His6 marcadas para a purificação de afinidade E. coli Seguido de cromatografia de troca aniônica, His6 tag-remoção com protease TEV e tamanho-exclusão cromatografia.

Vídeo: Purification of Hsp104, a Protein Disaggregase

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 the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purificação do M. magneticum Strain AMB-1 proteína associada Magnetosome MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Expressão, solubilização, detergente e Purificação de um transportador de membrana, as proteínas MexB resistência a múltiplas drogas

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Automated interação hidrofóbica Seleção coluna de cromatografia para uso na purificação de proteínas

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

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 ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Geração, purificação, caracterização e de Cell-invasivos espécies de proteína Disc1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

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 ...

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. ...