The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

1. Produção de plasmídeo e Shadoo Expressão Nota: O gene que codifica a proteína de murino Shadoo (Shadoo 25-122), foi subclonado no vector de expressão pET-28 11. Este clone foi usado para transformar E. SoluBL21 coli estirpe bacteriana, para expressar a proteína Shadoo com uma etiqueta de hexa-histidina N-terminal, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG), como explicado anteriormente 11. O plasmídeo pode ser solicitado dos autores. O peso molecular calculado a partir da Shadoo sua sequência de aminoácidos é 12,243.2 Da. Nota: Executar todas as manipulações com bactérias sob uma capela de fluxo laminal ou perto de Bunsen chama. <ol><li> Transformar bactérias competentes SoluBL21 com o pET-28-Sua 6 -Shadoo plasmídeo, usando um protocolo de choque térmico padrão. Para esta mistura de 1 a 5 ul do plasmídeo (tipicamente de 10 a 100 pgng) em 50 ul de as bactérias num tubo. Realizar a choque térmico a 42 ° C durante 45 seg. </li><li> Cultura de bactérias transformadas a 37 ° C até 600 um de 0,5-0,7 em meio de Luria Bertani suplementado com 40 mg / ml de canamicina (Figura 1B). </li><li> Shadoo induzir expressão O / N através da adição de 240 ug / ml de isopropil-β-D-tiogalactopiranósido, ITPG, para a cultura. As bactérias de cultura sob agitação a 150 rpm a 37 ° C. Nota: Normalmente, cultura bacteriana alcança A 600, de 3 de O / N. Nota: Transformado bactérias podem ser armazenadas a -80 ° C para a produção de proteína futuro. Para isso, adicionam-se 10% -50% (v / v) de glicerol estéril em alíquotas de culturas bacterianas e congelá-las a -80 ° C. </li></ol><img alt="figura 1" src="/files/ftp_upload/53432/53432fig1.jpg" /> Figura 1. Esquema de Shadoo construção de expressão, transformação de bactérias e de indução de expressão Shadoo (tal como descrito no passo 1). </strong&gt; (A) A construção de expressão para Shadoo codifica uma marca de hexa-histidina fundida com o N-terminal da proteína, Nt. O esquema mostra que a proteína contém repetições Shadoo básicos arginina / glicina (cilindro verde), um domínio hidrófobo (HD), e um único local de N-glicosilação (CHO), seguido por um terminal-C (TC). (B) 28 pET-6-His -Shadoo plasmídeo é transformado em bactérias SoluBL21 por choque térmico durante 45 segundos a 42 ° C. Bactérias transformadas são plaqueadas em placas de agar selectivo que continha 40 ug / ml de canamicina. Uma única colónia da placa é utilizada para inocular um cultura num frasco agitador de 1.000-3.000 ml. A expressão da proteína recombinante é induzida com IPTG 1 mM. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig1highres.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 2. Purificação de Proteínas Shadoo por Cromatografia Líquida de Baixa Pressão (LPLC) <ol><li> A lise celular e Emcorpos conclusão preparação <ol><li> Recolha a suspensão bacteriana e transferi-la para dentro de tubos de centrifugação (Figura 2). </li><li> Rotação a 2.500 xg, durante 20 min a 4 ° C. Remover o sobrenadante por decantação e pelotas ressuspender em 15 ml de tampão (Tris 50 mM, EDTA 10 mM, pH 8, com anti-proteases) para cada sedimento derivado de 500 ml de cultura de células. Nota: Os sedimentos ressuspensos podem ser congeladas e armazenadas a -20 ° C durante vários dias. Congelamento facilita a lise de células bacterianas. </li><li> Verifique a sobre-expressão de proteína recombinante utilizando bactérias peletes bruto por análise de SDS-PAGE e coloração com Coomassie. <ol><li> Para isso, de centrífuga de 1 ml de cultura bacteriana para sedimentar as células. Adicionar 100 ul de solução de desnaturação de Laemmli (277,8 mM de Tris-HCl, pH 6,8, 4,4% de SDS, 44,4% w / v de glicerol, 0,02% azul de bromofenol) para sedimentar e aquece-se a 100 ° C durante 5 min. </li><li> Carga de 10 ul da amostra num gel de acrilamida a 12%, executar SDS-PAGE e visuAlize proteínas separado por coloração com Coomassie. </li></ol></li><li> Adicionar Triton X-100 a 0,5% de concentração final para o sedimento completamente ressuspenso de 2.1.2). </li><li> Incubar a suspensão em banho de água a 37 ° C durante 30 min. </li><li> Sonicar a suspensão durante 5 minutos a 6-12 microns amplitude pico-a-pico em água gelada para lisar células bacterianas. </li><li> Transferência suspensão sonicada em 50 ml de tubos de centrifugação limpas. </li><li> Tubos de centrifugação a 15.000 xg durante 15 min a 4 ° C. </li><li> Remover o sobrenadante por decantação e adicionam-se 5 ml de tampão de ligação (tampão de 20 mM de Tris-HCl, pH 7,4, NaCl 0,5 M, Ureia 8 M, 5 mM de imidazole) por tubo. </li><li> Colocar os tubos na roda girando O / N para proteínas a partir de corpos de inclusão voltar a suspender completamente. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53432/53432fig2.jpg" /> Figura 2. Esquema de purificação de inclusãocorpos (tal como descrito no passo 2.1). transformadas que expressam Shadoo bactérias são colhidas por centrifugação e ressuspensas no tampão de lise. A suspensão é incubada a 37 ° C durante 30 min e, em seguida sonicada para romper mecanicamente células bacterianas. A sonicação é feito em gelo para evitar o aquecimento da amostra. Quebrados células são colhidas por centrifugação e ressuspensas num tampão contendo 8 M ureia para solubilizar proteínas a partir de corpos de inclusão. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig2highres.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> </li><li> Cromatografia em Ni 2+ -affinity Nota: A cromatograf ia é realizada no FPLC usando um Sistema de Ni 2+ -charged, 5 ml de Sepharose quelante (Figura 3A). Todas as soluções são carregados na coluna com um caudal de 1 mL / min. <ol><li> Injectar 10 ml de solução 4 M Niso água de 0,2 para a coluna de Sepharose, a fim de carregá-lo-ions com Ni 2+. </li><li> Equilibrar a coluna carregada com Ni 2+ -ions por injecção de 30-50 ml de tampão de ligação contendo uma baixa concentração de imidazolo (tampão de 20 mM de Tris-HCl, pH 7,4, NaCl 0,5 M, Ureia 8 M, 5 mM de imidazole). </li><li> Coloque as proteínas solubilizadas-ureia com um dispositivo de injecção de 50 ml. </li><li> Recuperar a fracção de fluxo mediante a lavagem da coluna com 10 ml de tampão de baixa concentração de imidazole de ligação (tampão Tris-HCl a 20, pH 7,4, 0,5 M de NaCl, 8 M de ureia, mM de imidazole 5), a fim de remover um máximo de proteínas não ligadas a partir da coluna. </li><li> Lava-se a coluna com 50-100 ml do tampão de lavagem (20 mM Tris-HCl, pH 7,4, NaCl 0,5 M, ureia 8 M, imidazol 80 mM) para eluir as proteínas não especificamente ligadas à coluna. </li><li> Aplicar um gradiente linear de 15 min de imidazolo 80-800 mM no tampão contendo 20 mM de Tris-HCl, pH 7,4, NaCl 0,5 M, Ureia 8 M. Recolher fracções de 1 ml durante a aplicação do gradiente. Nota: proteínas-His-tag são eluídos sagacidadegradiente ha de tampão contendo imidazole (utilizado como competidor). </li><li> Tomar uma alíquota de 8 mL de cada fração e adicione 4 mL solução de desnaturação 4x Laemmli (ver 2.1.3.1). Soluções de aquecer a 100 ° C durante 5 min. </li><li> Carga de 10 ul de marcador de tamanho de proteína e 10 ul de cada amostra para o gel de acrilamida a 12%, executar SDS-PAGE e visualizar as proteínas separadas por coloração com Coomassie. Nota: O peso molecular calculado de Shadoo formar a sua sequência de aminoácidos é 12,243.2 Da. </li><li> Piscina todas as fracções contendo Shadoo. </li></ol><img alt="Figura 3" src="/files/ftp_upload/53432/53432fig3.jpg" /> Figura 3. Procedimento de purificação (tal como descrito nos passos 2,2-2,4). (A) Ni2 + -ions pode ser coordenado por resíduos de histidina, que permite a purificação selectiva de proteínas marcadas com poli-histidina. Imidazole é utilizado para eluir a proteína, atravéssua capacidade para coordenar com o Ni 2+ -ion e deslocar a proteína ligada. (B) As proteínas de diferentes raio hidrodinâmico que foram retidas na Ni 2+ -column são separados numa coluna de exclusão de tamanho. As proteínas são eluídas a partir da maior para os mais pequenos. (C) antes de usar, as fracções contendo Shadoo são reunidas e dessalinizadas para remover a ureia, imidazol e os seus sais. Os ensaios de controlo de qualidade incluem SDS-PAGE / coloração com Coomassie, Western blotting. Purificada Shadoo pode ser liofilizado. Liofilizado Shadoo é estável durante meses, quando armazenadas a -20 ° C. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig3highres.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> </li><li> A cromatografia de exclusão de tamanho Nota: Um segundo passo de purificação consiste na cromatografia de exclusão de tamanho que separa proteína Shadoo das outras proteínas co-eluídas no primeiro passo de purificação(Figura 3B). <ol><li> Equilibrar coluna Superdex de 120 ml de volume total do leito através da injecção de 250 ml de tampão de 20 mM de Tris-HCl, pH 7,4, NaCl 0,5 M, Ureia 8 M a uma taxa de fluxo de 1,5 ml / min. </li><li> Carregar fracções reunidas contendo a partir de 2.2.9 Shadoo na coluna equilibrada. Recolher fracções de 1 ml após o volume vazio da coluna de 40 ml sobre o fluxo constante do tampão anterior. </li><li> Tomar uma aliquota de 10 ul de cada fracção e realização de uma análise de SDS-PAGE e coloração com Coomassie, tal como descrito acima para saber quais as fracções contêm Shadoo. </li><li> Piscina fracções contendo Shadoo. </li></ol></li><li> Dessalinização Nota: A dessalinização procedimento remove os sais, ureia e imidazole para se obter a proteína recombinante Shadoo num tampão adequado para estudos bioquímicos e biofísicos (Figura 3C). O mesmo resultado pode ser obtido por diálise contra o tampão de amostras. <ol><li> Equilibrar uma coluna de dessalinização de 53 ml com 150 mlde tampão de acetato de amónio 10 mM, pH 5, a uma taxa de fluxo de 5 ml / min. </li><li> Carga reunidas fracções Shadoo na coluna. Após a injecção do tampão acetato de amónio recolher manualmente fracções que mostram um aumento do sinal a 280 nm na saída do detector de UV. </li><li> Liofilizar a Shadoo dessalinizada e armazená-lo a -20 ° C. </li><li> Dissolver Shadoo liofilizado num tampão adequado, o tampão de acetato de sódio tal como 10 mM, pH 5, antes da utilização. Nota: A etiqueta de poli-histidina pode ser removida com enteroquinase. </li></ol></li></ol> 3. In Vitro Montagem dos Shadoo em amilóide fibrilas no pH fisiológico Nota: Os agregados proteicos Shadoo e espontaneamente forma fibrilhas a pH ≥ 7 11. <img alt="Figura 4" src="/files/ftp_upload/53432/53432fig4.jpg" /> Figura 4. Esquema da Shadoo redobragem para fibrilas amilóides (como descricama no passo 3). Purificada Shadoo converte-se espontaneamente para fibrilas amilóides quando dissolvido em tampão de pH 7. ≥ numa solução ácida, Shadoo converte em fibrilas amilóides após incubação com 1 M Gdn-HCl enquanto se agita. Os ensaios de controlo de qualidade incluem microscopia eletrônica e coloração TT. <a href="https://www.jove.com/files/ftp_upload/53432/53432fig4highres.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <ol><li> Diluir Shadoo para uma concentração final de 2 ^ M num tampão 20 mM Tris-HCl, pH 7,4. Medir a concentração, medindo a densidade óptica da solução a 280 nm e utilizando o coeficiente de extinção deduzida a partir da composição de aminoácidos Shadoo de 20.970. </li><li> Incubar 500 ul da solução em um tubo de plástico cónico a 4 ° C durante um par de semanas, para permitir a conversão de proteína espontânea de estruturas amilóides (Figura 4). </li><li> Add preparados na hora tioflavina T (TT) para um conce definitivantration de 10 uM a uma alíquota de 100 ul da solução Shadoo. Incubar a solução durante 5 min à TA. Medir emissão de fluorescência a partir de 460 a 520 nm, utilizando um comprimento de onda de excitação de 435 nm, para verificar a presença de estruturas amilóides 17. </li></ol> 4. In Vitro Montagem dos Shadoo para amilóide fibrilas no ácida pHs <ol><li> Diluir Shadoo em 1 M GdnHCl, tampão de 20 mM de acetato de Na, pH de 5 a uma concentração final de 2 ^ M. </li><li> Incubar 500 ul desta solução num tubo cónico com agitação contínua a 37 ° C, durante 3-7 dias. </li><li> Verifique a presença de fibrilhas amilóides por adição de TT a uma alíquota da solução como em 3.3. </li><li> Dialisar suspensão obtida contra tampão de 10 mM de acetato de Na, pH 5,0, para purificar Shadoo fibrilas amilóides. </li><li> Armazenar fibrilas purificada a 4 ° C. </li></ol><img alt="Figura 5" src="/files/ftp_upload/53432/53432fig5.jpg"/&gt; As fracções de proteína Figura 5. Os resultados representativos. Eluídas a partir da coluna carregada com Ni2 + quelante (A) e uma coluna de exclusão de tamanho (B) foram analisadas usando SDS-PAGE e coloração com Coomassie. Identificação de Shadoo purificada foi avaliada utilizando anticorpo anti-Shadoo SPRN (C). <a href="https://www.jove.com/files/ftp_upload/53432/53432fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<ol>
	<li>Biasini, E., Turnbaugh, J. A., Unterberger, U., Harris, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+at+the+crossroads+of+physiology+and+disease.">Prion protein at the crossroads of physiology and disease.</a> Trends in Neurosciences. 35, 92-103 (2012).</li><li>Colby, D. W., Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+generation+of+prion+strains.">De novo generation of prion strains.</a> Nature Reviews. Microbiology. 9, 771-777 (2011).</li><li>Chiti, F., Dobson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding,+functional+amyloid,+and+human+disease.">Protein misfolding, functional amyloid, and human disease.</a> Annual Review of Biochemistry. 75, 333-366 (2006).</li><li>Nelson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+cross-beta+spine+of+amyloid-like+fibrils.">Structure of the cross-beta spine of amyloid-like fibrils.</a> Nature. 435, 773-778 (2005).</li><li>Murphy, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+amyloid+formation+and+membrane+interaction+with+amyloidogenic+proteins.">Kinetics of amyloid formation and membrane interaction with amyloidogenic proteins.</a> Biochimica et Biophysica Acta. 1768, 1923-1934 (2007).</li><li>Steunou, S., Chich, J. F., Rezaei, H., Vidic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensing+of+lipid-prion+interactions:+insights+on+charge+effect,+Cu(II)-ions+binding+and+prion+oligomerization.">Biosensing of lipid-prion interactions: insights on charge effect, Cu(II)-ions binding and prion oligomerization.</a> Biosensors &amp; Bioelectronics. 26, 1399-1406 (2010).</li><li>Watts, J. C., Westaway, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prion+protein+family:+diversity,+rivalry,+and+dysfunction.">The prion protein family: diversity, rivalry, and dysfunction.</a> Biochimica et Biophysica Acta. 1772, 654-672 (2007).</li><li>Westaway, D., Daude, N., Wohlgemuth, S., Harrison, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PrP-like+proteins+Shadoo+and+Doppel.">The PrP-like proteins Shadoo and Doppel.</a> Topics in Current Chemistry. 305, 225-256 (2011).</li><li>Baillod, P., Garrec, J., Tavernelli, I., Rothlisberger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+versus+doppel+protein+misfolding:+new+insights+from+replica-exchange+molecular+dynamics+simulations.">Prion versus doppel protein misfolding: new insights from replica-exchange molecular dynamics simulations.</a> Biochemistry. 52, 8518-8526 (2013).</li><li>Daude, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+Shadoo+proteins+convert+to+amyloid-like+forms+under+native+conditions.">Wild-type Shadoo proteins convert to amyloid-like forms under native conditions.</a> Journal of Neurochemistry. 113, 92-104 (2010).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shadoo+binds+lipid+membranes+and+undergoes+aggregation+and+fibrillization.">Shadoo binds lipid membranes and undergoes aggregation and fibrillization.</a> Biochemical and Biophysical Research Communications. 438, 519-525 (2013).</li><li>Watts, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CNS+glycoprotein+Shadoo+has+PrP(C)-like+protective+properties+and+displays+reduced+levels+in+prion+infections.">The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections.</a> The EMBO Journal. 26, 4038-4050 (2007).</li><li>Watts, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-resistant+prions+selectively+decrease+Shadoo+protein.">Protease-resistant prions selectively decrease Shadoo protein.</a> PLoS Pathogens. 7, e1002382 (2011).</li><li>Westaway, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+Shadoo+in+prion+infections+traces+a+pre-clinical+event+inversely+related+to+PrP(Sc)+accumulation.">Down-regulation of Shadoo in prion infections traces a pre-clinical event inversely related to PrP(Sc) accumulation.</a> PLoS Pathogens. 7, e1002391 (2011).</li><li>Chevalier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PB1-F2+influenza+A+virus+protein+adopts+a+beta-sheet+conformation+and+forms+amyloid+fibers+in+membrane+environments.">PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.</a> The Journal of Biological Chemistry. 285, 13233-13243 (2010).</li><li>Tarus, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+paths+of+the+nucleoprotein+of+influenza+A+virus.">Oligomerization paths of the nucleoprotein of influenza A virus.</a> Biochimie. 94, 776-785 (2012).</li><li>Biancalana, M., Koide, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+Thioflavin-T+binding+to+amyloid+fibrils.">Molecular mechanism of Thioflavin-T binding to amyloid fibrils.</a> Biochimica et Biophysica Acta. 1804, 1405-1412 (2010).</li><li>Minic, J., Sautel, M., Salesse, R., Pajot-Augy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+system+as+a+screening+tool+for+pharmacological+assessment+of+g+protein+coupled+receptors.">Yeast system as a screening tool for pharmacological assessment of g protein coupled receptors.</a> Current Medicinal Chemistry. 12, 961-969 (2005).</li><li>Schein, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cool+way+to+make+proteins.">A cool way to make proteins.</a> Nature Biotechnology. 22, 826-827 (2004).</li><li>Sorensen, H. P., Mortensen, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+genetic+strategies+for+recombinant+protein+expression+in+Escherichia+coli.">Advanced genetic strategies for recombinant protein expression in Escherichia coli.</a> Journal of Biotechnology. 115, 113-128 (2005).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+glycosylation+enhances+ubiquitin-mediated+proteasomal+degradation+of+Shadoo+in+Scrapie-infected+rodents+and+cultured+cells.">Disruption of glycosylation enhances ubiquitin-mediated proteasomal degradation of Shadoo in Scrapie-infected rodents and cultured cells.</a> Molecular Neurobiology. 49, 1373-1384 (2014).</li><li>Englund, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+biosynthesis+of+glycosyl+phosphatidylinositol+protein+anchors.">The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors.</a> Annual Review of Biochemistry. 62, 121-138 (1993).</li><li>Puig, B., Altmeppen, H., Glatzel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GPI-anchoring+of+PrP:+implications+in+sorting+and+pathogenesis.">The GPI-anchoring of PrP: implications in sorting and pathogenesis.</a> Prion. 8, 11-18 (2014).</li></ol>

The authors have nothing to disclose.

Um protocolo de fácil e eficiente tanto para expressão e purificação de uma grande quantidade de proteína recombinante de ratinho Shadoo é apresentado. O método descrito permite a solubilização e purificação bem sucedida de (His 6) tagged Shadoo proteína recombinante. É importante, tal como referido no título, que quando expresso em uma bactéria procariota E. Shadoo coli acumula-se em corpos de inclusão. Antes da purificação Shadoo, as bactérias têm que ser lisadas por so...

Doenças neurodegenerativas causadas por príons, que incluem a encefalopatia espongiforme bovina em bovinos, scrapie em ovinos ea doença de Creutzfeldt-Jakob em humanos, são fatal e incurável. Prion doenças são caracterizadas por mudanças conformacionais da proteína príon celular composta principalmente de hélices, no-β-sheet cruz enriquecido amilóide conformer 1,2. Cross-beta-estruturas contêm densamente embalado e beta-folhas-cristalinos como altamente ordenadas estabilizado com ligações de hidrogênio 3,4. Amyloids Príon se auto-replicar devido aos beta-fios na borda crescente que fornecem um modelo para recrutar e converter uma unidade de proteína monomérica. De acordo com a hipótese do &quot;única proteína&quot;, o conformador de proteína amilóide prião é o agente infeccioso único. No entanto, algumas outras biomoléculas têm sido propostos como sendo essencial para distúrbios do prião. Por exemplo, as membranas celulares são pensados ​​para ser um lugar onde conversiocorre e em alguns lípidos carregados negativamente têm sido mostrados para melhorar o processo de conversão de 5,6. Além disso, algumas proteínas também podem estar envolvidos em patologias prião. Especificamente, existem dois outros membros da família de proteínas prião: Duplo e Shadoo 7,8. Doppel tem uma estrutura relativamente rígida estabilizado com pontes de di-sulfureto e não autopolimerização, e agregada para fibrilas amilóides 9. Em contraste, semelhante à da proteína do prião, Shadoo pode adoptar uma estrutura β folhas enriquecido e auto-associar-se em estruturas de fibrilas amilóides. Foi demonstrado que pode Shadoo fibrilar em condições nativas ou sobre as membranas carregadas negativamente 10,11 ligação. Conversão de Shadoo em fibras de amilóide-like pode ser associada com patologias. Com efeito, os mecanismos pelos quais as estruturas amilóides como causam a doença são mal compreendidos. Shadoo tem um domínio hidrofóbico (HD) e uma série de conjunto Arg / Gly repete t semelhanteO a parte N-terminal da proteína do prião (Figura 1A). À medida que a parte N-terminal da proteína do prião, Shadoo é altamente carregada positivamente e parece ser uma proteína nativa desestruturado 11,12. Shadoo parece ser funcionalmente relacionados com distúrbios do prião, uma vez que se pode ligar directamente a proteína prião. Além disso, a sua expressão é regulada negativamente durante prião patologia 13,14. No entanto, o papel de Shadoo na doença do prião ainda não foi estabelecido. Foi desenvolvido um plasmídeo que transporta a sequência de codificação do gene de rato Shadoo. O plasmídeo foi usado para transformar E. coli para a produção de N-terminal His Shadoo 6 fusão. Este sistema de expressão é bem estabelecida em nosso laboratório e é comumente usado em nossos projetos em andamento 6,15,16. Um problema importante para a expressão de Shadoo é a escolha da E. estirpe de E. coli. Enquanto BL21 competente estirpe bacteriana é geralmente utilizado para a expressão de pró recombinanteteínas Shadoo foi expressa e purificada a partir da estirpe única bacterianas transformadas com sucesso SoluBL21. SoluBL21 E. competente coli é um mutante melhorada da estirpe BL21 de acolhimento desenvolvido para a produção de proteínas cuja expressão na estirpe BL21-mãe deu nenhum produto detectável solúvel. Expressão de alto nível de Shadoo em SoluBL21 E. coli conduz a acumulação da proteína em corpos de inclusão. Como uma característica geral, quando uma proteína não nativa é altamente expressa em E. coli, esta proteína tende a acumular-se em corpos de inclusão insolúveis. Shadoo agrega provavelmente através de interacções não covalentes ou iónicas hidrofóbicas (ou uma combinação de ambos) para estruturas dinâmicas altamente enriquecidos formadas pela proteína dobrada para vários graus. Em consequência, a purificação compreende pelo menos dois passos: (i) uma separação selectiva da proteína a partir de outras biomoléculas em um meio de desnaturação, e (ii) uma renaturação da proteína purificada utilizando na dobragem in vitrotécnicas. A separação selectiva de Shadoo foi conseguida numa solução tampão contendo ureia 8M (ou, alternativamente, 6M guanidina-HCl). A remoção da ureia e a renaturação da proteína pode ser realizada por aplicação de vários protocolos de: (i) renaturação em uma solução de pH ácido para se obter uma proteína Shadoo monomérica desestruturado, ou (ii) renaturação em uma solução de pH ≥ 7 para se obter Shadoo polimerizado em fibras de amilóide estáveis ​​com motivos característicos cross-β-folha.

<table>
	<tbody>
		<tr>
			<td>AKTA FPLC&#160;</td>
			<td>GE, Amersham</td>
			<td># 1363</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/60 Superdex</td>
			<td>GE Healthcare</td>
			<td>17-1069-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap IMAC</td>
			<td>GE Healthcare</td>
			<td>GE17-0920-05</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep26/10 Desalting column</td>
			<td>GE Healthcare</td>
			<td>GE17-5087-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SoluBL21 Competent E. coli</td>
			<td>Genlantis</td>
			<td>C700200</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-28b+</td>
			<td>Novogen</td>
			<td>69865-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth medium</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Biophotometar</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td></td>
		</tr>
		<tr>
			<td>Trito X-100</td>
			<td>Life Technologies</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>NiSO4</td>
			<td>Sigma-Aldrich</td>
			<td>656895</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Gdn-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr>
			<td>protein size marker&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

O sistema de expressão de Escherichia coli é uma ferramenta poderosa para a produção de proteínas eucarióticas recombinantes. Nós usá-lo para produzir Shadoo, uma proteína pertencente à família de prião. Um método de cromatografia para a purificação de (His) 6 tagged Shadoo recombinante expressa como corpos de inclusão é descrito. Os corpos de inclusão solubilizados em ureia 8 M e ligado a uma coluna de Ni 2+ -charged para realizar cromatografia de afinidade de iões. As proteínas ligadas são eluidas com um gradiente de imidazole. As fracções contendo proteína Shadoo são submetidas a cromatografia de exclusão de tamanho para obter uma proteína altamente purificada. No passo final é purificado Shadoo dessalinizada para remover os sais, ureia e imidazole. Shadoo proteína recombinante é um reagente importante para estudos bioquímicos e biofísicos de distúrbios conformação da proteína que ocorrem nas doenças de priões. Muitos relatórios demonstraram que as doenças neurodegenerativas priões são originários da deposição de facadale, ordenou fibrilas amilóides. Protocolos de exemplo que descrevem como fibrilar Shadoo em fibrilas amilóides no ácido e neutro / pHs básicos são apresentados. Os métodos de como produzir e fibrilar Shadoo pode facilitar a investigação em laboratórios que trabalham com doenças de príon, uma vez que permite a produção de grandes quantidades de proteína de uma maneira rápida e de baixo custo.

Cerca de 5-10 mg de proteína Shadoo purificado é obtido por litro de cultura bacteriana. A purificação em duas fases é necessário para obter Shadoo recombinante de elevado grau de pureza. A primeira etapa é realizada por cromatografia de afinidade com uma coluna de Ni 2+ -charged que conserva as suas-proteínas marcadas. As fracções eluídas são sujeitas a SDS-PAGE e corados com Coomassie Brilliant Blue (Figura 5A). As fracções que contêm proteína são reunidas e Shadoo adiciona...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Purificação e redobramento de amilóide fibrilas de (seu)<sub&gt; 6</sub&gt; tagged proteína recombinante Shadoo Expresso como Corpos de Inclusão em<em&gt; E. coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

purification-refolding-to-amyloid-fibrils-his6-tagged-recombinant

Research

JoVE Journal

Biology

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

14.1K visualizações.  INRA. A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures. 

Vídeo: Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Gravação de patch-clamp de canais iônicos Expresso em Oócitos Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Biologia

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 ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagênese e Funcional Protocolos de Seleção para a Evolução Dirigida de proteínas em E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

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 Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

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

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Ensaios para Monitoramento juntamente redobramento de proteínas em<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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;

Purificação da fibrose cística condutância transmembrana Regulador proteína expressa em<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening expressão quantitativa e Purificação Aplicada à recombinante Dissulfeto ricos em proteínas veneno produzido em<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

A purificação cromatográfica do não-polipéptidos recombinantes Elastina semelhantes e suas fusões com Peptídeos e Proteínas de<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...