Reconocemos el Dr. Amir Aharoni por su apoyo y Geula Davidov, Grimberg Noam y Guttman Chen por sus consejos y comentarios.

1. Clonación y expresión de genes en E. mamá coli El gen mutante mamAΔ41 se amplificó mediante la reacción en cadena de la polimerasa (PCR) de ADN genómico de Magnetospirillum magneticum AMB-1, con los cebadores: 5&#39;-GCATTACGCATATGGACGACATCCGCCAGGTG-3 &#39;y 5&#39;-GCGCGGCAGCCATA-TGGCATACG-3&#39;. En los fragmentos de ADN amplificados, un sitio NcoI se introdujo en el codón de iniciación ATG y el codón de terminación fue sustituido por un sitio de SCOI. Los fragmentos fueron digeridos con NcoI y SacI y se clonó en los respectivos sitios de pET52b (+), dando lugar a pET52bMamAΔ41-AMB1. En esta construcción, el gen mamAΔ41 se fusionó en marco con el Su 10-etiqueta en el C-terminal. El plásmido se electroporación en la cepa de E. coli BL21. Cepa de E. coli BL21 albergar pET52bMamAΔ41 se cultivó en auto-inducción (15) un medio que contiene ampicilina (50 mg / ml) 310 ° K durante 3 horas. La temperatura de cultivo fue cambiado luego de 310 ° a 300 ° K y mantenido durante 48 horas a 300 ° K. Las células fueron cosechadas por centrifugación a 5465g durante 10 minutos a 277 ° K. 8 litros cultura produce 60 gramos de celda húmeda de pellets. 2. Bioinformática cálculos Los cálculos de peso molecular (MW), de acuerdo a la secuencia de aminoácidos y la absorción prevista de 1mg/1ml de proteína en 280 nm, utilizando el servidor ProtParam (http://www.expasy.ch/tools/protparam.html). Para 10His-Tag-MamAΔ41 el MW es 22.529 Da (240 aminoácidos) y de MamAΔ41 (con 9 aminoácidos izquierda después de su etiqueta de eliminación de la trombina) es el Mw 20596.5Da (187 aminoácidos). La absorción prevista de 1mg/1ml de proteína en 280 nm para 10His-Tag-MamAΔ41 es 0,595 y 0,579 para MamAΔ41 es. Además, la secuencia de aminoácidos no contiene residuos de cisteína. Por lo tanto, agentes reductores no son necesarios durante el proceso de purificación. 3. La purificación de MamAΔ41 <ol><li> De preparación de la solución, pesar y preparar (200 ml cada una) en las siguientes concentraciones: imidazol (228,2 MW g / mol) solución de 4M, NaCl (Mw 58,44 g / mol) solución 5 M, Tris-HCl (121,3 Mw g / mol solución) 1 M, ajustar el pH = 8. Agregue el agua doblemente destilada (DDW) y se mezcla con un vórtex hasta que la solución es clara. Soluciones de filtro con filtro de 0.22μm y mantener a temperatura ambiente. </li><li> Preparar y filtro 6 buffers frescos de las soluciones madre (Tabla 1). </li><li> Diluir y suspender las células en un buffer de volumen en relación 1:2, los gramos de células en el búfer (ml). </li><li> Añadir 10μl para cada 10gr de bacterias de DNasa I (1 mg / ml) a fin de romper los fragmentos de ADN en la muestra. Añadir 1 ml por cada 20gr de bacterias de EDTA cóctel de inhibidores de la proteasa libre. Incubar durante 20 min en hielo. </li><li> Interrumpió las células por dos ciclos de la prensa francesa de 25.000 psi. A diferencia de ultrasonidos, la prensa francesa está dirigida a un volumen alto (10 ml&gt;) de extrusión celular al forzar a las células a alta presión a través de un orificio delgado. El pistón debe ser enfriada en hielo durante diez minutos antes de su uso y montaje debido a la acumulación de temperatura. </li><li> Transferencia de las células lisadas en tubos de 25 ml de ultracentrifugación y el equilibrio con precisión (50 mg) con un buffer hasta que la muestra llega a la línea del tubo s cuello. </li><li> Restos de células separadas por ultracentrifugación a 270.000 g durante 1 hora a 277 ° K. </li><li> Prepare una gravedad casera Ni NTA columna (2,5 cm de diámetro) mediante la adición de 4 ml de Ni NTA resina, que se conserva en el 20% de etanol, en la columna. Dejó caer el líquido por completo, lavar con 40 ml de DDW y dejó caer el líquido por completo. Pre-equilibrio de la resina en la columna con 40 ml de tampón A. </li><li> Aplicar la fracción soluble de los tubos de ultracentrifugación de la gravedad Ni columna NTA. Dejó caer el líquido, recoger el flujo a través de (proteínas libre) y preservar a 277K. </li><li> Recoja la muestra de sedimento de los tubos ultra-centrifugación mediante el uso de una pequeña propina para el desguace de la pastilla. Mezclar la punta en 50μl de 5% β-mercaptoetanol en 2X SDS-PAGE muestra de amortiguación. </li><li> Lavar la columna con 100 ml de tampón B. Que la caída de líquidos, recoger el filtrado y conservar a 277K. </li><li> Prepare 15 tubos eppendorf marcados (1,5 ml). </li><li> Elución - válvula de columna cerrar y volver a la carga de 1 ml de solución tampón en la columna C, se incuba durante 3 min seguido de la apertura de la válvula y la recolección de flujo continuo. Repita este paso tres veces. </li><li> Eluir resto de la suspensión en 1 ml de búfer incrementos de C y cerrar la válvula. </li><li> Medida de OD a 280 nm de las fracciones y evaluar la ubicación del pico de proteína, si la densidad óptica es aún&gt; 0,3, las muestras se necesitan. </li><li> Análisis SDS-PAGE de las fracciones recogidas. Dicho análisis debe llevarse a cabo también con los siguientes ejemplos, las proteínas no consolidados a través de flujo, lavado de corriente y de uARLT-centrifugación pellet de la muestra. Mezcla de 10μl de cada muestra con 10μl de 5% β-mercaptoetanol en 2X SDS-PAGE muestra de amortiguación. Cargar las muestras, el 15% SDS-PAGE y una duración de 45 minutos en 180 voltios. </li><li> Tinción del gel en 15 ml de solución de InstantBlue, agitar durante 1 hora. Asegúrese de que la caja de plástico está cubierto y protegido de la luz. </li><li> Evaluar gel, si la expresión de proteínas es alta y específica de acuerdo con las bandas de indicativo, combinación seleccionado fracciones eluidas. </li><li> Con el fin de eliminar de 10 Su-etiqueta, añadir la trombina bovina (10 unidades / l), 10μl de trombina por 1 mg de proteína, de acuerdo con las recomendaciones del fabricante para la muestra combinada. </li><li> La diálisis se utiliza para eliminar el exceso de imidazol y disminuir las concentraciones de NaCl de los niveles necesarios durante el atascamiento del ion del cambio. Para la diálisis, cortar la longitud deseada de 7 kD de peso molecular corte (MWCO) tubos de diálisis. Lavar con DDW y hacer un nudo en un extremo (o un sello con clips). Transferencia de solución de proteína se fusionó en la bolsa, dejando algo de espacio en la parte superior y abrazadera para arriba. </li><li> Sumerja el tubo de diálisis en tampón de diálisis 4 litros refrigerados (Buffer D). Agitar la solución lentamente durante la noche a 277 ° K. </li><li> Saque el tubo de diálisis del vaso, hace una pequeña incisión y la transferencia de la proteína de 50 ml de tubo. </li><li> Cromatografía de intercambio iónico: reunir preenvasados ​​MonoQ 4.6/100 columna PE en cromatografía líquida rápida de rendimiento (FPLC). Lavar la columna con 5 volúmenes de columna (CV) de agua filtrada DDW. Equilibra la columna con 5 VC de buffer D. </li><li> Lave el circuito de inyección con DDW (dos volúmenes de bucle) y repetir con el tampón de carga D. la proteína en el circuito de inyección. </li><li> Inyectar la muestra de proteínas (velocidad de flujo de 1 ml / min) y se empezarán a recoger las fracciones para la detección de SDS-PAGE de las proteínas sin límites. </li><li> Si el volumen inicial de proteína (paso 3,23) es mayor que el circuito de inyección, repita los pasos 3.25-3.26, sin lavar el circuito, hasta que toda la muestra se carga en la columna. </li><li> Lavar la columna con 3 CV de tampón C con el fin de eliminar las proteínas sin límites. </li><li> Eluir la proteína con un gradiente lineal de 60 minutos de 40 2000mm NaCl entre topes D y E (cuando el 100% E buffer es el final de la pendiente), la tasa de flujo de 1 ml / min (a pesar de mayores tasas de flujo se puede utilizar en el costo de volúmenes más grandes, pero menos concentrado, los picos de proteínas). Recoger los picos de proteínas que aparecen en el cromatograma de 280 nm. </li><li> Limpie preenvasados ​​columna de acuerdo para la fabricación de recomendación. </li><li> Análisis SDS-PAGE de las fracciones recogidas. Dicho análisis debe realizarse con los siguientes ejemplos, las proteínas sin límites de flujo continuo y proteínas picos de flujo a través de. Ejecute el SDS-PAGE y tinción como menciona en los pasos 3.17-3.18. </li><li> Evaluar gel de proteínas en la existencia de bandas Mw previsto de acuerdo con la proteína pre-manchada marcador. Combinación seleccionado fracciones de elución que contienen la proteína correspondiente. </li><li> Diálisis de las muestras combinadas, a fin de reducir las concentraciones de NaCl necesaria para cromatografía de exclusión. Realizar la diálisis como se menciona en los pasos 3,21-3,23 frente a tampón F. </li><li> Concentrar la muestra de la proteína usando un Vivaspin-15 10000 MWCO a 8 mg / ml en una centrífuga de mesa, 4.000 rpm. Validar la concentración de deseo mediante la medición de OD a 280 nm en cubetas de cuarzo. Si la proteína está muy concentrada que se diluye con tampón F y vuelva a medir. </li><li> Ensamble de exclusión de tamaño preenvasados ​​columna, HiLoad 26/60 Superdex 200, en el FPLC, lavar la columna con 3 CV de agua filtrada DDW. Equilibra la columna con 3 CV de tampón F. </li><li> Lave bucle de inyección de 5 ml con DDW (dos volúmenes de bucle), seguido por el mismo volumen con el tampón de carga F. no más de 4 ml de la muestra de proteínas en el circuito de inyección. </li><li> Inyectar la muestra de proteínas (velocidad de flujo de 3,5 ml / min) y se empezarán a recoger las fracciones pico de proteínas que aparecen es el cromatograma de 280 nm. Deja que el flujo de buffer hasta que se alcanza un volumen de columna. </li><li> Si el volumen inicial de proteína (paso 3,23) es mayor que el circuito de inyección, repita los pasos 3.25-3.26, sin lavar el circuito, hasta que toda la muestra se carga en la columna. </li><li> Limpie preenvasados ​​columna de acuerdo con la recomendación de la fabricación. </li><li> Análisis SDS-PAGE de las fracciones recogidas. Dicho análisis debe llevarse a cabo con los picos de proteínas de muestras. Ejecute el SDS-PAGE y tinción como menciona en los pasos 3.17-3.18. </li><li> Evaluar gel: si los picos de proteínas específicas, en el Mw correcta previsto de acuerdo con la proteína pre-manchada marcador y sólo es visible una banda de combinación seleccionado fracciones eluidas de la proteína correspondiente. </li><li> Concentrar la muestra de proteínas con un Vivaspin-15 10000 MWCO a&gt; 20 mg / ml en una centrífuga de mesa, 4.000 rpm. Validar la concentración de deseo mediante la medición de OD a 280 nm. El MamAΔ41 purificada se concentró a 26,5 mg / ml para la cristalización. </li> &lt;li&gt; Confirmar pureza de la muestra y la identificación de proteínas utilizando Matrix asistida por láser de desorción / ionización (MALDI-TOF). </li><li> Si la proteína es purificada de acuerdo con el espectrómetro de masas y SDS-PAGE, se divide el MamAΔ41 concentrado para pre-marcadas tubos Eppendorf, 25-50μl de proteínas en cada uno. </li><li> Flash congelado en nitrógeno líquido y se almacena a 193 ° K. </li></ol> 4. Resultados representante Cuando este protocolo se hace correctamente se debe tener muy purificada, el tamaño de las muestras de proteínas homogénea y concentrada. Estas muestras de proteínas están listas para los ensayos de cristalización, así como estudios bioquímicos, tales como la cinética de enzimas, la afinidad de unión y mucho más. Aquí descritos son resultados representativos de los pasos principales en el protocolo de purificación. SDS-PAGE análisis del perfil de elución de Ni-NTA columna de afinidad que revelan muy a las proteínas expresadas en la fracción soluble en MW apropiado de ~ 22kDa (Figura 1). Este análisis SDS-PAGE también debe revelar mínimo si cualquier proteína en las proteínas sin límites a través del flujo, lavar el filtrado y ultra-centrifugación de la muestra de pellets. Si las bandas grandes de la proteína adecuada no aparecen en estas muestras se debe considerar la preparación de búfer incorrecto, los problemas de ruptura celular o problemas en la cultura de inducción auto-condiciones y el crecimiento. Cromatografía de intercambio iónico está preformada para separar entre nuestra proteína deseable para otras proteínas de E. coli que se unido a la resina de níquel (debido a las interacciones electrostáticas o histidina / negativo lazos de aminoácidos ricos en proteínas) y sin cortes de etiquetado Su proteína deseable. Esta columna separa las proteínas de acuerdo a su afinidad de unión a la resina con carga positiva en las concentraciones crecientes de NaCl. Proteínas de alta carga negativa se eluyen en mayores concentraciones de NaCl aproximar a los moderados de carga negativa. Las ventajas de esta columna son de alto caudal y capacidad de unión. El cromatograma de intercambio iónico (Figura 2) muestra una buena separación entre las tres poblaciones de proteínas en la creciente concentración de NaCl. SDS-PAGE se necesita un análisis para determinar MW de cada población, aislando a la deseable y la evaluación de si otras medidas de purificación son necesarios. La primera población es MamAΔ41 (~ 20 kDa), mientras que la segunda población es MamAΔ41 + Su Tag (~ 22kDa) que no se separó de la trombina bovina y la tercera población es indetectable en SDS-PAGE, debido a la baja concentración. Si los picos de las proteínas no se separan claramente se debe considerar la preparación de búfer incorrecto o el cambio de la pendiente del gradiente de NaCl. Cromatografía de exclusión está preformada para separar entre nuestra proteína deseable para otras proteínas de las células de E. coli que se unido a la resina Ni-NTA y fueron separados durante cromatografía de intercambio iónico. Esta columna separa las proteínas según su tamaño. Proteínas de gran tamaño se eluyen en el volumen de elución más pequeños frente a los pequeños que se eluye en grandes volúmenes. El cromatograma de la columna (Figura 3) muestra una buena separación de la proteína deseable como una población principal. La población parece eluir en el tamaño adecuado de monómero con un peso molecular de ~ 20 kDa. La presencia de la banda de ~ 80 kDa (proteína de E.coli típicos que se une la resina Ni-NTA) se detecta en SDS-PAGE antes de la carga de columna y desaparece durante esta carrera. Dado que la concentración de esta proteína ~ 80 kDa es baja para empezar, su población no aparece en el cromatograma, debido a su dilución y SDS-PAGE se necesita un análisis para determinar la eficiencia de depuración. Recomendamos cargar una muestra diluida y concentrada de la cumbre principal de la SDS-PAGE para un seguro podría determinar la presencia de una proteína pura en el MW apropiado. En esta etapa de la proteína se purifica y su homogeneidad deben ser evaluadas por MALDI-TOF. Este análisis reveló una proteína en MW de 20.347 Da y otro en Mw de 10.176 Da (Figura 4). El ~ 10 kDa es el ~ 20 kDa duplicado cargos. El MW previstos de MamAΔ41 con los 9 aminoácidos izquierda después de su etiqueta de eliminación fue 20596,5 Da. Al compararla con la obtenida MALDI-TOF MW se encontró que son 248 Da aparte. Esta diferencia puede deberse a errores de medición MALDI-TOF y / o debido a la degradación común de la primera metionina y glicina el segundo. Para concluir, la proteína es altamente purificada y se puede utilizar para otros experimentos. <img src="/files/ftp_upload/1844/1844table.jpg" alt="Tabla 1" /> Tabla 1. Formulaciones de búfer. <img src="/files/ftp_upload/1844/1844fig1.jpg" alt="Figura 1" /> Figura 1. Representante SDS-PAGE análisis de purificación en columna Ni-NTA De derecha a izquierda;. P-ultra-centrífuga de pellets (3,11 paso del protocolo), W-lavado (3,12 paso del protocolo), U-sin límites proteínas (3,10 paso del protocolo), E- cinco carriles de elución samples (3,14 paso del protocolo), M - marcador de la proteína (números indican MW). La flecha indica MamAΔ41. <img src="/files/ftp_upload/1844/1844fig2.jpg" alt="Figura 2" /> Figura 2. El análisis de cromatografía de intercambio iónico paso (a) El intercambio de iones (columna MonoQ) cromatograma, azul - 280 nm de absorción, representa la concentración de proteínas, Green-NaCl de concentración, Rojo - indicación de las fracciones recogidas. (B) Representante de SDS-PAGE análisis de la etapa de purificación de intercambio iónico. De izquierda a derecha, M - marcador de la proteína (números indican Mw), A6 - fracción primer pico, A8-segunda fracción pico. <img src="/files/ftp_upload/1844/1844fig3.jpg" alt="Figura 3" /> Figura 3. El análisis por cromatografía de exclusión por tamaño (a) la exclusión de tamaño (columna Superdex 200) cromatograma, azul - 280 nm de absorción, representa la concentración de proteínas, la indicación de Red de las fracciones recogidas. (B) Representante de SDS-PAGE análisis de la etapa de purificación por exclusión de tamaño. De izquierda a derecha, M-proteína marcadora (los números indican MW), muestra de PREI-pre-inyección, A4-6 fracción combinada recogidos en el primer pico, A4-6D-diluida fracción recogida desde el pico. <img src="/files/ftp_upload/1844/1844fig4.jpg" alt="Figura 4" /> Figura 4. Láser asistida por matriz de desorción / ionización (MALDI-TOF) espectro de masas de agua purificada MamAΔ41. La matriz es el ácido sinápico (SA). MamAΔ41 muestra a 20.347 Da, también se muestra es la especie de doble carga de MamAΔ41 a 10.176 Da.

<ol>
	<li>Faivre, D., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+Bacteria+and+Magnetosomes.">Magnetotactic Bacteria and Magnetosomes.</a> Chem Rev. 108, 4875-4898 (2008).</li><li>D'Andrea, L. D., Regan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TPR+proteins:+the+versatile+helix.">TPR proteins: the versatile helix.</a> Trends Biochem Sci. 28, 655-662 (2003).</li><li>Young, J. C., Barral, J. M., Hartl, U. l. r. i. c. h., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+folding:+localized+functions+of+cytosolic+chaperones.">More than folding: localized functions of cytosolic chaperones.</a> Trends Biochem Sci. 28, 541-547 (2003).</li><li>Brocard, C., Hartig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+targeting+signal+1:+is+it+really+a+simple+tripeptide?.">Peroxisome targeting signal 1: is it really a simple tripeptide?.</a> Biochim Biophys Acta. 1763, 1565-1573 (2006).</li><li>Fransen, M., Amery, L., Hartig, A., Brees, C., Rabijns, A., Mannaerts, G. P., Van Veldhoven, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+PTS1-+and+Rab8b-binding+properties+of+Pex5p+and+Pex5Rp/TRIP8b.">Comparison of the PTS1- and Rab8b-binding properties of Pex5p and Pex5Rp/TRIP8b.</a> Biochim Biophys Acta. 1783, 864-873 (2008).</li><li>Baker, M. J., Frazier, A. E., Gulbis, J. M., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein-import+machinery:+correlating+structure+with+function.">Mitochondrial protein-import machinery: correlating structure with function.</a> Trends Cell Biol. 17, 456-464 (2007).</li><li>Mirus, O., Bionda, T., von Haeseler, A., Schleiff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionarily+evolved+discriminators+in+the+3-TPR+domain+of+the+Toc64+family+involved+in+protein+translocation+at+the+outer+membrane+of+chloroplasts+and+mitochondria.">Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria.</a> J Mol Model. 15, 971-982 (2009).</li><li>Gatsos, X., Perry, A. J., Anwari, K., Dolezal, P., Wolynec, P. P., Likic, V. A., Purcell, A. W., Buchanan, S. K., Lithgow, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+secretion+and+outer+membrane+assembly+in+Alphaproteobacteria.">Protein secretion and outer membrane assembly in Alphaproteobacteria.</a> FEMS Microbiol Rev. 32, 995-1009 (2008).</li><li>Tiwari, D., Singh, R. K., Goswami, K., Verma, S. K., Prakash, B., Nandicoori, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+residues+in+Mycobacterium+tuberculosis+protein+kinase+G+play+a+role+in+regulating+kinase+activity+and+survival+in+the+host.">Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.</a> J Biol Chem. 284, 27467-27479 (2009).</li><li>Edqvist, P. J., Broms, J. E., Betts, H. J., Forsberg, A., Pallen, M. J., Francis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetratricopeptide+repeats+in+the+type+III+secretion+chaperone,+LcrH:+their+role+in+substrate+binding+and+secretion.">Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.</a> Mol Microbiol. 59, 31-44 (2006).</li><li>Grunberg, K., Muller, E. C., Otto, A., Reszka, R., Linder, D., Kube, M., Reinhardt, R., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+proteomic+analysis+of+the+magnetosome+membrane+in+Magnetospirillum+gryphiswaldense.">Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense.</a> Appl Environ Microbiol. 70, 1040-1050 (2004).</li><li>Komeili, A., Vali, H., Beveridge, T. J., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosome+vesicles+are+present+before+magnetite+formation,+and+MamA+is+required+for+their+activation.">Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation.</a> Proc Natl Acad Sci USA. 101, 3839-3843 (2004).</li><li>Okuda, Y., 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=Expression+and+characterization+of+a+magnetosome-associated+protein,+TPR-containing+MAM22,+in+Escherichia+coli.">Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli.</a> FEBS Lett. 491, 169-173 (2001).</li><li>Taoka, A., Asada, R., Sasaki, H., Anzawa, K., Wu, L. 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>

Purificación de proteínas es el paso principal en todas las proteínas bioquímicos o estudios estructurales. Dado que cada proteína es única con su propio comportamiento, es necesario definir sus propiedades y modificar en consecuencia su purificación. Blanco de la proteína debe ser analizado como un primer paso hacia la purificación usando herramientas bioinformáticas. Que se utilizan para calcular el objetivo de punto isoeléctrico, evaluar su necesidad de la reducción / oxidación medio ambiente y su necesi...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<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

Las bacterias magnetotácticas comprenden un grupo heterogéneo de microorganismos acuáticos que son capaces de orientarse a lo largo de los campos geomagnéticos. Este comportamiento se cree para ayudar a su búsqueda de ambientes adecuados<em&gt; (1)</em&gt;. Esta capacidad es conferido por la magnetosomas, un orgánulo subcelular que consta de un conjunto de cadena lineal de las vesículas de lípidos cada uno capaz de biomineralize y adjuntar un ~ 50 nm de cristales de magnetita o greigite. Un componente principal de la magnetosomas que ha demostrado ser necesarios para la formación de vesículas funcional es mamá. Mamá es una muy abundante magnetosomas proteína asociada a que es uno de los más caracterizados magnetosomas proteínas asociadas<em&gt; In vivo</em&gt;<em&gt; (2-6)</em&gt;. Este artículo se centra en la purificación de la mamá, que a pesar de ser estudiado<em&gt; In vivo</em&gt;, No hay detalles funcionales o estructurales han sido identificados por él. Bioinformática análisis sugiere que mamá es una repetición de tetra-tricopeptide (TPR), que contiene proteínas. TPR es un motivo estructural encontrado como parte o formación de un pliegue más importante en un amplio rango de proteínas, sirve como un modelo para las interacciones proteína-proteína y proteína interviene en varios complejos<em&gt; (7)</em&gt;. EPC están implicados en muchas tareas cruciales en la célula eucariota procesos orgánulo y muchas vías de bacterias<em&gt; (8-14).</em&gt; Para entender Mama, a TPR única que contiene la proteína, la proteína altamente purificada se requiere como un primer paso. En este artículo se presenta el protocolo de purificación de un mutante estable Mama eliminación (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 M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Mamá es una única proteína magnetosomas asociados, que ha demostrado ser involucrados en la activación de magnetosomas. Aquí se presenta el protocolo de purificación de Mama supresión mutante (MamAΔ41) de<em&gt; M. magneticum</em&gt; AMB-1.

La purificación de la M. magneticum Tensión AMB-1 magnetosomas proteína asociada 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

13.5K vistas.  Ben-Gurion University. Mamá es una única proteína magnetosomas asociados, que ha demostrado ser involucrados en la activación de magnetosomas. Aquí se presenta el protocolo de purificación de Mama supresión mutante (MamAΔ41) de M. magneticum AMB-1.

Video: Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41

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.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

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

Investigación

Biología

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.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

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

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificación de los progenitores del músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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.

La interacción hidrofóbica automatizada cromatografía en columna de selección para uso en la purificación 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.

La purificación de Hsp104, una proteína Disaggregase

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.

Identificación de los socios interacción de proteínas en tándem de purificación de afinidad

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.

Expresión y purificación de la proteína transmembrana de la fibrosis quística regulador de la conductancia en la 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.

Florecimiento extracción de proteínas de levadura y purificación para el Estudio de la función, las interacciones y modificaciones post-traduccionales

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.

Identificación de modificaciones post-traduccionales de la proteína vegetal Complejos

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;