Reconhecemos Dr. Amir Aharoni por seu apoio e Davidov Geula, Noam Grimberg e Chen Guttman para os seus conselhos e comentários.

1. Clonagem e expressão de Mama no Gene E. coli O mutante do gene foi amplificado mamAΔ41 usando a reação em cadeia da polimerase (PCR) de DNA genômico de Magnetospirillum magneticum AMB-1, com primers: 5&#39;-GCATTACGCATATGGACGACATCCGCCAGGTG-3 &#39;e 5&#39;-GCGCGGCAGCCATA-TGGCATACG-3&#39;. Nos fragmentos de DNA amplificado, um site NcoI foi introduzido no códon de iniciação ATG eo códon de terminação foi substituído por um site SCOI. Os fragmentos foram digeridos com NcoI e SacI e clonados no respectivos sites de pET52b (+), dando origem a pET52bMamAΔ41-AMB1. Nesta construção, o gene mamAΔ41 foi fundida em-frame com o 10-Sua tag no C-terminal. O plasmídeo foi electroporated em E. coli estirpe BL21. E. coli estirpe BL21 abrigar pET52bMamAΔ41 foi cultivado em auto-indução (15) meio contendo ampicilina (50 mg / ml) 310 ° K por 3 horas. A temperatura de cultivo foi então deslocada de 310 ° a 300 ° K e mantido por um adicional de 48 h, a 300 ° K. As células foram colhidas por centrifugação a 5465g por 10 minutos a 277 ° K. 8 cultura de litros produzidos 60 gramas de pellet celular molhado. 2. Os cálculos de bioinformática Cálculos de peso molecular (MW), de acordo com a seqüência de aminoácidos ea absorção previsto de 1mg/1ml de proteína em 280nm, utilizando o servidor ProtParam (http://www.expasy.ch/tools/protparam.html). Para 10His-Tag-MamAΔ41 o MW é 22529 Da (240 aminoácidos) e para MamAΔ41 (com 9 aminoácidos esquerda após sua remoção por tag-trombina) o Mw é 20596.5Da (187 aminoácidos). A absorção previsto de 1mg/1ml de proteína em 280nm para 10His-Tag-MamAΔ41 é 0,595 e para MamAΔ41 é 0,579. Além disso, a seqüência de aminoácidos não contém resíduos de cisteína. Portanto, agentes redutores não são necessários durante o processo de purificação. 3. Purificação da MamAΔ41 <ol><li> Para a preparação de solução estoque, pesar e preparar (200 ml cada) nas seguintes concentrações: Imidazol (MW 228,2 g / mol) solução 4M, NaCl (Mw 58,44 g / mol) solução 5M, Tris-HCl (Mw 121,3 g / mol ) solução 1M, ajustar o pH = 8. Adicionar água destilada duas vezes (DDW) e misture usando um vortex até que a solução é clara. Soluções de filtro com filtro 0.22μm e manter em temperatura ambiente. </li><li> Prepare e filtrar 6 buffers frescos a partir de soluções estoque (Tabela 1). </li><li> Diluir e suspender as células em tampão Um volume em 1:2 ratio, gramas de células para buffer (ml). </li><li> Adicionar 10μl para cada 10gr de bactérias de DNase I (1 mg / ml) a fim de quebrar fragmentos de DNA na amostra. Adicionar 1 ml para cada 20gr de bactérias de EDTA coquetel inibidor de protease livre. Incubar por 20 min no gelo. </li><li> Interrompido células por dois ciclos de imprensa francesa a 25.000 psi. Ao contrário de sonicação, a imprensa francesa tem como objetivo em alto volume (10ml&gt;) extrusão de células, forçando as células sob alta pressão através de um orifício fino. O pistão deve ser resfriado em gelo por 10 minutos antes do seu uso e de montagem devido ao acúmulo de temperatura. </li><li> Transferência de células lisadas em tubos de ultracentrífuga 25ml e equilibrá-los com precisão (50mg) com tampão A até que a amostra atinge a linha de tubo s pescoço. </li><li> Restos celulares separado por centrifugação a 270.000 g Ultra por 1 hora a 277 ° K. </li><li> Prepare um caseiro gravidade Ni coluna NTA (2,5 cm de diâmetro), adicionando 4 ml de Ni NTA, que são preserva em etanol 20%, na coluna. Deixe o líquido cair completamente, em seguida, lavar com 40 ml de DDW e deixe o líquido cair completamente. Pré-equilibrada a resina na coluna com 40 ml de tampão A. </li><li> Aplicar a fração solúvel de ultra-centrifugação de tubos sobre a gravidade coluna NTA Ni. Deixe o líquido cair, recolher flow-through (proteínas não ligada) e preservar a 277k. </li><li> Coletar amostra de pellet dos tubos de ultracentrifugação usando uma pequena dica para acabar com o pellet. Misture a ponta em 50μl de 5% β-mercaptoetanol em SDS-PAGE 2X tampão de amostra. </li><li> Lavar a coluna com 100 ml de tampão B. Deixe a queda de líquidos, coleta de fluxo e preservar a 277k. </li><li> Prepare 15 tubos eppendorf marcados (1,5 ml). </li><li> Eluição - Válvula coluna próxima e então carregar 1ml de tampão C na coluna; incubar por 3 min seguido de abertura da válvula ea vazão através da recolha. Repita este passo três vezes. </li><li> Eluir resto de suspensão de 1 ml incrementos tampão C e fechar a válvula. </li><li> OD medida em 280nm das frações e avaliar localização do pico de proteína, se o OD ainda é&gt; 0,3, as amostras adicionais são necessários. </li><li> SDS-PAGE análise das frações coletadas. Tal análise deve ser realizada também com os exemplos a seguir, as proteínas unbound flow-through, lavagem de fluxo e ultra-centrifugação da amostra de sedimento. Mix 10μl de cada amostra com 10μl de 5% β-mercaptoetanol em SDS-PAGE 2X tampão de amostra. Coloque as amostras em 15% SDS-PAGE e correr por 45 min em 180 Volts. </li><li> Manchar o gel em 15 ml de solução InstantBlue, agitar durante 1 hora. Certifique-se que a caixa de plástico é coberto e protegido da luz. </li><li> Avaliar gel, se a expressão da proteína é elevado e específico de acordo com as faixas indicativas, mesclagem selecionado frações eluídas. </li><li> A fim de remover 10-Seu-tag, adicionar trombina bovina (10 unidades / mL), 10μl de trombina por 1 mg de proteína, de acordo com a recomendação do fabricante para a amostra combinada. </li><li> A diálise é utilizada para a remoção de excesso de Imidazol e para diminuir as concentrações de NaCl para os níveis necessários durante a ligação de íons de câmbio. Para diálise, corte comprimento desejado, de 7 de peso molecular de corte kD (MWCO) tubos de diálise. Lavar com DDW e faça um nó em uma extremidade (ou selo com clipes). Transferência de solução de proteína fundiram no saco, deixando algum espaço na parte superior e grampo para cima. </li><li> Tubo de submergir a diálise em 4 tampão de diálise Litros refrigerados (buffer D). Agitar a solução lentamente durante a noite com 277 ° K. </li><li> Tire o tubo de diálise do copo, faça um pequeno corte e transferência da proteína para 50ml tubo. </li><li> Cromatografia de troca iônica: montar pré-embalados MonoQ 4.6/100 coluna PE em Cromatografia Rápida Desempenho líquido (FPLC). Lavar a coluna com 5 volumes coluna (CV) de água filtrada DDW. A coluna equilibrada com 5 CV de tampão D. </li><li> Lavar o loop de injeção com DDW (dois volumes loop) e repita com tampão D. Load a proteína no circuito de injeção. </li><li> Injectar a amostra de proteínas (vazão de 1 ml / min) e iniciar a coleta de frações de SDS-PAGE detecção de proteínas sem limites. </li><li> Se o volume inicial de proteína (passo 3.23) é maior do ciclo de injeção, repita os passos de 3,25-3,26, sem lavar o loop, até que toda a amostra é carregada na coluna. </li><li> Lavar a coluna com 3 CV de tampão C, a fim de eliminar as proteínas sem limites. </li><li> Eluir a proteína com um gradiente linear de 60 min 40 2000mm NaCl entre buffers D &amp; E (E quando buffer de 100% é o fim do gradiente), o fluxo de taxa de 1 ml / min (embora maiores taxas de fluxo pode ser usado no custo de volumes maiores, mas menos concentrado picos, proteína). Coletar picos de proteínas como eles aparecem no cromatograma 280nm. </li><li> Coluna de pré-embalados de acordo limpa para a fabricação de recomendação. </li><li> SDS-PAGE análise das frações coletadas. Tal análise deve ser realizada com as seguintes amostras, as proteínas ilimitada de fluxo e proteína picos de fluxo contínuo. Executar o SDS-PAGE e mancha como falar em passos de 3,17-3,18. </li><li> Avaliar gel para a existência de bandas de proteínas na Mw previsto de acordo com a proteína marcador pré-manchado. Mesclar frações de eluição selecionadas que contêm a proteína em questão. </li><li> Diálise das amostras fundidas, a fim de reduzir as concentrações de NaCl necessária para cromatografia por exclusão de tamanho. Realizar a diálise, como mencionado nos passos 3,21-3,23 contra tampão F. </li><li> Concentrar a amostra de proteína usando um Vivaspin-15 10000 MWCO a 8 mg / ml em uma centrífuga de mesa, 4000 rpm. Validar concentração desejo medindo OD em 280nm em cubetas de quartzo. Se a proteína é muito concentrada diluir com tampão F e medir novamente. </li><li> Montar tamanho exclusão coluna pré-embalados, HiLoad 26/60 Superdex 200, em FPLC; lavar a coluna com 3 CV de água filtrada DDW. A coluna equilibrada com 3 CV de tampão F. </li><li> Lavar ciclo de injeção com 5ml DDW (dois volumes loop) seguiu pelo mesmo volume com tampão F. Não coloque mais de 4 ml da amostra de proteína no ciclo de injeção. </li><li> Injectar a amostra de proteínas (vazão de 3,5 ml / min) e iniciar a coleta de frações de proteínas de pico como eles aparecem se o cromatograma 280nm. Deixe o fluxo de buffer até atingir o volume da coluna. </li><li> Se o volume inicial de proteína (passo 3.23) é maior do ciclo de injeção, repita os passos de 3,25-3,26, sem lavar o loop, até que toda a amostra é carregada na coluna. </li><li> Coluna de pré-embalados limpo de acordo com a recomendação de fabricação. </li><li> SDS-PAGE análise das frações coletadas. Tal análise deve ser realizada com picos nas amostras de proteínas. Executar o SDS-PAGE e mancha como falar em passos de 3,17-3,18. </li><li> Avaliar gel: se picos de proteínas são específicas, na Mw correta previsto de acordo com a proteína marcador pré-coradas e apenas uma banda é visível mesclagem selecionado frações eluídas da proteína em questão. </li><li> Concentre-se amostra de proteína usando um Vivaspin-15 10000 MWCO a&gt; 20 mg / ml em uma centrífuga de mesa, 4000 rpm. Validar concentração desejo medindo OD em 280nm. O MamAΔ41 purificada foi então concentrada a 26,5 mg / ml para a cristalização. </li> &lt;li&gt; Confirme a pureza da amostra e identificação de proteínas usando Matrix-assisted laser de dessorção / ionização (MALDI-TOF). </li><li> Se a proteína é purificada de acordo com o espectrômetro de massa e SDS-PAGE, divida o MamAΔ41 concentrado para tubos eppendorf pré-marcada, 25-50μl de proteína em cada um. </li><li> Flash congelados em nitrogênio líquido e armazenar a 193 ° K. </li></ol> 4. Resultados representante Quando este protocolo é feito corretamente deve-se altamente purificado, tamanho amostras de proteína homogênea e concentrada. Estas amostras de proteínas são, então, pronto para ensaios de cristalização, bem como estudos bioquímicos, tais como cinética enzimática, afinidade de ligação e muito mais. Aqui descritos são resultados representativos dos principais passos no protocolo de purificação. SDS-PAGE análise do perfil de eluição de Ni-NTA coluna de afinidade deve revelar muito mais proteína expressa na fração solúvel em MW apropriado de ~ 22kDa (Figura 1). Esta análise SDS-PAGE deve também revelar mínimo, se qualquer proteína nas proteínas ilimitada de fluxo, lavagem de fluxo e ultra-centrifugação da amostra de sedimento. Se grandes bandas da proteína apropriada aparecem nestas amostras deve-se considerar a preparação de buffer errado, problemas no rompimento celular ou problemas na cultura de auto-indução condições e crescimento. Cromatografia de troca iônica é pré-formados a fim de separar entre o nosso proteína desejável outras proteínas E.coli que estavam vinculados à resina de níquel (devido a interações eletrostáticas ou histidina / negativo aminoácidos loops ricos em proteínas) e proteína His-Tagged uncut desejável. Esta coluna separa proteínas de acordo com a afinidade de ligação à resina com carga positiva no aumento da concentração de NaCl. Proteína altamente carregadas negativamente vai eluir em maiores concentrações de NaCl appose para moderar os com carga negativa. As vantagens desta coluna são de alta vazão e capacidade de ligação. O cromatograma de troca iônica (Figura 2) revela uma boa separação entre 3 populações proteínas no aumento da concentração de NaCl. SDS-PAGE análise é necessária a fim de determinar MW de cada população, isolando a uma desejável e avaliar se as etapas de purificação que são necessários mais. A primeira população é MamAΔ41 (~ 20 kDa), enquanto a segunda população é MamAΔ41 + Tag His (~ 22kDa) que não foi clivada pela trombina bovina e à terceira população é indetectável no SDS-PAGE, devido à baixa concentração. Se os picos de proteínas não são claramente separados deve-se considerar a preparação de buffer errado ou alterar a inclinação do gradiente de NaCl. Cromatografia por exclusão de tamanho é pré-formados a fim de separar entre o nosso proteína desejável a outras proteínas de células E. coli que foram ligados à resina Ni-NTA e não foram separados durante a cromatografia de troca iônica. Esta coluna separa proteínas de acordo com seu tamanho. Grandes proteínas vai eluir em menores volumes de eluição oposição a pequenos que serão eluídos em volumes maiores. O cromatograma coluna (Figura 3) revela uma boa separação da proteína desejável como uma população principal. A população parece eluir no tamanho apropriado com um monômero MW de ~ 20 kDa. A presença de ~ 80 kDa banda (E.coli proteína típica que se liga Ni-NTA) é detectado em SDS-PAGE antes de carregá-coluna e desaparece durante essa rodada. Desde que a concentração desta proteína kDa ~ 80 é baixo, para começar, a sua população não aparece no cromatograma, devido à sua diluição e SDS-PAGE análise é necessária para determinar a eficiência de purificação. Recomendamos o carregamento de uma amostra diluída e concentrada do pico principal do SDS-PAGE para um certamente poderia determinar a presença de uma proteína pura no MW apropriado. Por esta fase, a proteína é purificada e sua homogeneidade deve ser avaliada por MALDI-TOF. Esta análise revelou uma proteína de 20.347 MW e outra em Da Mw de 10176 Da (Figura 4). O ~ proteína de 10 kDa é o ~ proteína kDa 20 dobrou cobrado. O MW previsto de MamAΔ41 com os 9 aminoácidos esquerda após sua Tag-remoção foi 20596,5 Da. Comparando-o com o obtido MALDI-TOF MW descobrimos que eles são 248 Da distante. Essa diferença pode ser resultado de erros MALDI-TOF medição e / ou devido à degradação comum do metionina primeiro eo segundo glicina. Para concluir, a proteína é altamente purificada e pode ser utilizado nos experimentos posteriores. <img src="/files/ftp_upload/1844/1844table.jpg" alt="Tabela 1" /> Tabela 1. Formulações Buffer. <img src="/files/ftp_upload/1844/1844fig1.jpg" alt="Figura 1" /> Figura 1. Representante análise SDS-PAGE de Ni-NTA purificação coluna Da direita para a esquerda;. P-ultra-centrifugação pellet (3.11 passo protocol), W-lavagem (3,12 etapa protocol), U-unbounded proteínas (3.10 passo protocol), E- cinco pistas de eluição samples (3,14 etapa protocol), M - marcador de proteína (números indicam MW). Seta indica MamAΔ41. <img src="/files/ftp_upload/1844/1844fig2.jpg" alt="Figura 2" /> Figura 2. Análise de cromatografia de troca iônica etapa (a) de troca iónica cromatograma (coluna MonoQ); Blue - 280nm absorção, representa a concentração de proteína, Green-concentração de NaCl, Vermelho - indicação das frações coletadas. (B) Representante análise SDS-PAGE da etapa de purificação de troca iônica. Da esquerda para a direita; M - marcador de proteína (números indicam Mw), A6 - fração primeiro pico, segundo A8-fração de pico. <img src="/files/ftp_upload/1844/1844fig3.jpg" alt="Figura 3" /> Figura 3. Tamanho análise de cromatografia de exclusão (a) a exclusão Tamanho (Superdex coluna 200) cromatograma; Blue - 280nm absorção, representa a concentração de proteína, a indicação Red das frações coletadas. (B) Representante análise SDS-PAGE do tamanho do passo de purificação de exclusão. Da esquerda para a direita; proteína M-marcador (números indicam MW), Prei pré-injeção de amostra, A4-6 fração combinada coletados a partir do primeiro pico, A4-6D-diluído fracção recolhida, a partir do pico. <img src="/files/ftp_upload/1844/1844fig4.jpg" alt="Figura 4" /> Figura 4. Matrix-assisted laser de dessorção / ionização (MALDI-TOF) espectro de massa de purificada MamAΔ41. A matriz é o ácido Sinapic (SA). MamAΔ41 mostrado em 20347 Da, também mostrado é a espécie dobrou acusado de MamAΔ41 em 10176 Da.

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F., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localizations+of+Mam22+and+Mam12+in+the+magnetosomes+of+Magnetospirillum+magnetotacticum.">Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum.</a> J Bacteriol. 188, 3805-3812 (2006).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expression and Purification. 41, 207-234 (2005).</li></ol>

Purificação de proteínas é o passo principal em todas as proteínas estruturais ou estudos bioquímicos. Uma vez que cada proteína é o único com seu próprio comportamento, é preciso definir suas propriedades e modificar sua purificação de acordo. Proteína-alvo deve ser analisado como um primeiro passo para a purificação utilizando ferramentas de bioinformática. Eles são usados ​​para calcular o objectivo ponto iso-elétrico, avaliar a sua necessidade de redução / oxidação ambiente e sua necessida...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French Press</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-078A</td>
<td>&nbsp;</td>
<tr>
<td>Pressure cell</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-032</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge</td>
<td>Equipment</td>
<td>Sorvall</td>
<td>Discovery 90SE</td>
<td>&nbsp;</td>
<tr>
<td>Rottor</td>
<td>Equipment</td>
<td>Beckman</td>
<td>Ti60</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml</td>
<td>Equipment</td>
<td>Beckman</td>
<td>BC-355618</td>
<td>&nbsp;</td>
<tr>
<td>2.5cm diameter, Glass Econo-Column Chromatography Columns</td>
<td>Equipment</td>
<td>BioRad</td>
<td>737-2521</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA His Bind resin</td>
<td>Equipment</td>
<td>Novagen</td>
<td>M0063428</td>
<td>&nbsp;</td>
<tr>
<td>Spectrophotometer</td>
<td>Equipment</td>
<td>Amersham Biosiences</td>
<td>Ultraspec 2100 pro</td>
<td>&nbsp;</td>
<tr>
<td>Quartz cuvette</td>
<td>Equipment</td>
<td>Hellma</td>
<td>104-QS</td>
<td>&nbsp;</td>
<tr>
<td>Fast Performance Liquid Chromatography- AKTA purifier 10</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>28-4062-64</td>
<td>&nbsp;</td>
<tr>
<td>Ion exchange column &#x2013; MonoQ 4.6/100 PE</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>10025543</td>
<td>&nbsp;</td>
<tr>
<td>Size exclusion pre-packed column-HiLoad 26/60 Superdex 200</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>17-1071-01</td>
<td>&nbsp;</td>
<tr>
<td>Centricon - Vivaspin15 &#x2013; 10,000 MWCO</td>
<td>Equipment</td>
<td>Sartorius Stedim Biotech GmbH</td>
<td>VS1501</td>
<td>&nbsp;</td>
<tr>
<td>Table centrifuge</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>IEC CL30R</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>Equipment</td>
<td>Bruker Daltonics</td>
<td>Reflex IV</td>
<td>&nbsp;</td>
<tr>
<td>Tris-HCl (hydrotymethyl) aminomethane</td>
<td>Reagent</td>
<td>BioLab</td>
<td>20092391</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>Reagent</td>
<td>FRUTROM</td>
<td>235553470</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole</td>
<td>Reagent</td>
<td>Alfa Aesar</td>
<td>288-32-4</td>
<td>&nbsp;</td>
<tr>
<td>EDTA free protease inhibitors cocktail</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-8849</td>
<td>&nbsp;</td>
<tr>
<td>Dnase I (Deoxyribonuclease I)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>DN-25</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Thrombin</td>
<td>Reagent</td>
<td>Fisher BioReagents</td>
<td>BP25432</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>BioLab</td>
<td>07132391</td>
<td>&nbsp;</td>
<tr>
<td>Soudim Dodecyl Sulfate (SDS)</td>
<td>Reagent</td>
<td>BioLab</td>
<td>19822391</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-3148</td>
<td>&nbsp;</td>
<tr>
<td>InstantBlue</td>
<td>Reagent</td>
<td>Expedeon</td>
<td>1SB01L</td>
<td>&nbsp;</td>
<tr>
<td>PageRuler Prestained Protein Ladder</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>SM0671</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

purification of the m. magneticum strain amb-1 magnetosome associated protein mama&delta;41

Bactérias magnetotáticas compreendem um grupo diverso de microorganismos aquáticos que são capazes de se orientar ao longo campos geomagnético. Este comportamento é acreditado para ajudar sua busca por ambientes adequados<em&gt; (1)</em&gt;. Esta capacidade é conferida pelo magnetosome, uma organela subcelulares que consiste de um conjunto linear de cadeia de lipídios vesículas cada um capaz de biomineralize e juntar um cristal ~ 50 nm de magnetita ou greigite. Um componente princípio da magnetosome que mostrou-se necessário para a formação de vesículas funcional é mamãe. Mama é uma proteína muito abundantes magnetosome-associados, que é um dos mais caracterizados magnetosome proteínas associadas<em&gt; In vivo</em&gt;<em&gt; (2-6)</em&gt;. Este artigo incide sobre a purificação da Mama, que apesar de ser estudado<em&gt; In vivo</em&gt;, Sem detalhes claros funcionais ou estruturais têm sido identificados por ele. Bioinformática análise sugeriu que mamãe é uma repetição tetra-tricopeptide (TPR), contendo proteína. TPR é um motivo estrutural encontrada como parte ou como formação de uma grande dobra em uma ampla gama de proteínas, ela serve como um modelo para interações proteína-proteína e proteína-mediador de vários complexos<em&gt; (7)</em&gt;. TPRs estão envolvidos em muitas tarefas cruciais nos processos de célula eucariótica organela e muitos caminhos bacteriana<em&gt; (8-14).</em&gt; Para entender Mama, um TPR única contendo proteína, proteína altamente purificada é necessária como um primeiro passo. Neste artigo, apresentamos o protocolo de purificação para um estábulo Mama exclusão mutante (MamAΔ41) de<em&gt; M. magneticum</em&gt; AMB-1.

Watch this Scientific Journal Video about Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÎ”41 at JoVE.com

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

Mama é uma proteína Magnetosome único associado que foi mostrado para ser envolvido na ativação magnetosome. Aqui apresentamos o protocolo de purificação da Mama exclusão mutante (MamAΔ41) de<em&gt; M. magneticum</em&gt; 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 ...

purification-m-magneticum-strain-amb-1-magnetosome-associated-protein

Research

JoVE Journal

Biology

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

13.4K visualizações.  Ben-Gurion University. Mama é uma proteína Magnetosome único associado que foi mostrado para ser envolvido na ativação magnetosome. Aqui apresentamos o protocolo de purificação da Mama exclusão mutante (MamAΔ41) de M. magneticum AMB-1.

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

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery Fase Líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

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 ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expressão e purificação do Fibrose Cística proteína transmembranar regulador da condutância na Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Brotando Yeast extração e purificação de proteínas para o estudo da função, interações e modificações pós-traducionais

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificação de modificações pós-translacionais de Complexos de proteína vegetal

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;