Este proyecto fue financiado por el Knut y Alice Wallenberg Foundation y el Consejo Sueco de Investigación.

1. La clonación de la fusión objetivo gene/ABDz1-tagged construir <ol><li> Preparar los fragmentos de PCR del gen ABDz1 flanqueado por sitios de restricción adecuados para N-terminal de la ligadura de la meta de genes en el plásmido de expresión (un plásmido que contiene ABDz1 está disponible gratuitamente a través de un acuerdo de transferencia de material). </li><li> Cleave purificada vector de expresión y los fragmentos de PCR con enzimas de restricción elegido en un tampón de reacción adecuado. Purificar los productos antes de la ligadura. </li><li> Ligar la restringida ABDz1 fragmento en el vector de expresión que contiene el gen de interés y transformar el producto de la ligadura de E. coli (en el método original de la cepa RR1ΔM15 se utilizó 8). Difundir las células transformadas en placas de agar suplementado con los antibióticos adecuados para la selección. </li><li> PCR pantalla de algunas colonias y la secuencia de verificar el cassette de expresión resultante de la secuenciación del ADN. </li><li> Preparar el plásmido de un cultivo de una noche de una secuencia de verifcolonia de IED y transformar a la cepa de expresión preferido (en el original método de E. coli Rosetta (DE3), recibiendo un pRARE plásmido para mejorar la producción de proteínas codificadas por los genes humanos, se utilizaron). </li></ol> 2. Expresión de la proteína <ol><li> Inocular una colonia de bacterias individuales en 10 ml de caldo de soja tríptico suplementado con extracto de levadura 5 g / l, y los antibióticos adecuados. Se incuba a 150 rpm a 37 ° C durante la noche. </li><li> Inocular 1 ml de cultivo de una noche en 100 ml de medio fresco e inducir la expresión de la proteína (originalmente 1 mM de isopropil-β-D-tiogalactósido cuando un operón lac se utilizó), cuando las células alcanzan la fase de crecimiento logarítmico. </li><li> Se incuba a 150 rpm a 25 ° C durante la noche las células y la cosecha por centrifugación. </li></ol> 3. Purificación por afinidad ortogonal <ol><li> Resuspender el precipitado en 25 ml de buffer en ejecución (25 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0,05% (w / v) de Tween 20, pH 8,0) e interrumpir ªlas células e por ultrasonidos a 60% de la amplitud y 1.0/1.0 pulsos durante 3 min. Centrifugar la muestra y el filtro de la proteína diana que contiene sobrenadante (0,45 m) antes de la purificación adicional. </li><li> Equilibrar un 1 ml de HSA Sepharose o una columna de NHS-activa, con anterioridad, junto con HSA de acuerdo con las recomendaciones del proveedor, con 10 volúmenes de columna (CV) de funcionamiento de amortiguación a 1 ml / min en un sistema de purificación de proteínas adecuada. Cargue el lisado bacteriano a 0,5 ml / min y luego restablecer el caudal de 1 ml / min. </li><li> Lavar la columna con 5 CV de funcionamiento de amortiguación, seguido de 5 CV de tampón de lavado (5 mM NH 4 Ac, pH 5,5). </li><li> Eluir la muestra con el tampón de elución (0,5 M HAc, pH 2,5) a 1 ml / min y recoger las fracciones, la absorción del monitor a 280 nm para seleccionar las fracciones del pico eluido para una mayor purificación. </li><li> Piscina de las fracciones con mayor concentración de proteínas, diluir dos veces en tampón de Claro en ejecución (fosfato 20 mM, NaCl 150 mM, pH 7,2) yasegúrese de que el pH es de alrededor de neutral. Añadir 1 M Tris-HCl pH 8 para aumentar el pH si es necesario. </li><li> Cargue la muestra de 0,5 ml / min en una columna de 1 ml MabSelect HiTrap Seguro que ha sido equilibrada con 10 CV de tampón Claro funcionando a 1 ml / min. Restablecer el caudal de 1 ml / min después de la carga. </li><li> Lavar la columna con 5 VC de tampón de Seguro (paso 5) y eluir las proteínas con 0,2 M de HAc, pH 2,7. De las proteínas diana sensibles que el efluente puede ser neutralizado directamente sobre la colección mediante la adición de Tris-HCl. </li></ol> Si los pasos de cromatografía se invierte el efluente de la columna MabSelect Claro que se puede diluir en la gestión de buffer (paso 3.1) y el pH aumentó a alrededor del 8 por adición de 1 M Tris-HCl. En general, se observan picos más estrechos cuando la matriz de Claro se emplea en la segunda etapa. 4. Evaluación de la pureza por electroforesis en gel de sodio dodecil sulfato de poliacrilamida (SDS-PAGE) <ol><li> Cargar las fracciones purificadas en unala reducción de SDS-PAGE y, si lo desea, analizar el peso molecular del producto purificado por espectrometría de masas. Es importante para reducir completamente la muestra, ya una cisteína libre en ABDz1 puede causar la dimerización de los productos en condiciones no reductoras. </li></ol> 5. Los resultados representativos: Como una prueba de principio, la purificación de afinidad ortogonal facilitado por la etiqueta ABDz1 se evaluaron durante tres proteínas diana humana que representan las diferentes clases de solubilidad, pesos moleculares y puntos isoeléctricos (Tabla 1). El gen ABDz1 fue fusionado genéticamente con los genes diana y las construcciones se expresaron en E. coli, la Figura 2 muestra un diagrama de flujo para todas las etapas del método de forma consecutiva. Después de la purificación por el protocolo ortogonales, las muestras recogidas en diferentes puntos fueron analizados por SDS-PAGE (Figura 3). Los resultados muestran claramente la utilidad de una etiqueta de doble y dos pasos de purificación muy específicos. A pesar de que la pureza es razonable achieved después de la etapa de purificación inicial como se ve desde los geles, el paso sucesivo se obtiene un producto muy puro muy adecuado para aplicaciones muy exigentes. <img alt="Figura 1" src="/files/ftp_upload/3370/3370fig1.jpg" /> Figura 1. Diseño de la biblioteca combinatoria utiliza para la selección de la biespecíficos ABDz1 etiqueta. Los 46 aminoácidos de unión a la albúmina pliegues de dominio en un conjunto estable de tres hélice y contiene un sitio de unión de la albúmina sérica humana, principalmente ubicados en la segunda hélice. Por genética aleatoria once superficie expuesta aminoácidos situada en la hélice de primera y tercera, una superficie de unión novela fue diseñado. Los once posiciones se indican en la figura y numeradas de acuerdo con Kraulis et al. 9. Después de biopanning contra un dímero de la Z-dominio de la proteína A estafilocócica con la biblioteca combinatoria expresadas en fagos, el biespecíficos ABDz1 molécula fue identificada. <img alt = &quot;Figura 2&quot; src = &quot;/ files/ftp_upload/3370/3370fig2.jpg&quot; /&gt; Figura 2. Un diagrama de flujo simplificado para el protocolo de purificación de afinidad ortogonal. Un fragmento de PCR que contiene la secuencia ABDz1 se liga (N-terminal) con un gen de interés en un vector de expresión adecuado. La ligadura de producto se transforma en E. coli para la verificación de la secuencia y la preparación de plásmidos. Después de la transformación de un huésped de la expresión, un cultivo a gran escala para la expresión de la proteína recombinante se configura a partir de un cultivo de una noche inicial. Las bacterias se recogieron por centrifugación y se lisaron por sonicación para producir lisado bacteriano que contiene la proteína diana. Después de una etapa de filtración para eliminar los restos de partículas sólidas, el lisado se somete a purificación por afinidad ortogonal en un sistema de manejo de líquidos al someterse a dos pasos de la purificación en la sucesión. Picos eluidos de los pasos de purificación se muestrean y se evaluó por SDS-PAGE para evaluar la pureza y en comparación con el tque lisado antes de la purificación. <img alt="Figura 3" src="/files/ftp_upload/3370/3370fig3.jpg" /> Figura 3. SDS-PAGE análisis de las proteínas objetivo expresado y purificado en la fusión con ABDz1. Las muestras tomadas del lisado bacteriano de tres proteínas que se expresan en la fusión de ABDz1, en representación de diferentes propiedades, y la etiqueta de ABDz1 sí fueron recogidos, junto con muestras de los picos de la purificación en una columna de HSA-seguido de un MabSelect Sure-columna (A) . Carriles 1-4 representan muestras de lisados ​​antes de la purificación, carriles 5-8 de la HSA, aguas (primer paso) y carriles de 9.12 de la purificación MabSelect Claro (segunda etapa). Las muestras se cargan en el siguiente orden: ABDz1-141377, ABDz1-HT875, ABDz1-HT2375 y ABDz1. Además, las muestras adquiridas a partir de los lisados ​​mismo purificado en el orden inverso en las mismas columnas se analizaron (B). Carriles 1-3 representan muestras de lisados ​​antes de la purificación, carriles de 4.6 from MabSelect Claro, aguas (primer paso) y los carriles 7-9 de HSA, aguas (segundo paso). Las muestras se cargaron en el mismo orden que en (A) pero la etiqueta no está incluido. A partir de esos resultados está claro que este método de dos pasos produce proteínas altamente purificadas, independientemente del orden en que las medidas se aplican. <table border="1"><tbody><tr><td> Nombre b </td><td> Proteína diana Uniprot c </td><td> Molecular peso (kDa) </td><td> Solubilidad clase d </td><td> Peso molecular del producto de la fusión (kDa) </td><td> Punto isoeléctrico del producto de la fusión </td></tr><tr><td> 141377 </td><td> B7Z315 </td><td> 17.4 </td><td> 4 </td><td> 23.7 </td><td> 8.5 </td></tr><tr><td> HT875 </td><td> P01040 </td><td> 10.8 </td><td> 3 </td><td> 17.1 </td><td> 4.9 </td></tr><tr><td> HT2375 </td><td> P00740 </td><td> 8.9 </td><td> 5 </td><td> 15.2 </td><td> 6.8 </td></tr></tbody></table><ol class="lalpha"><li> Peso molecular de ABDz1 etiqueta sólo es de 6,3 kDa, punto isoeléctrico 6,7. </li><li> Proteína diana representa una parte de la proteína Uniprot. </li><li> <a href="http://www.uniprot.org">http://www.uniprot.org</a> . </li><li> Solubilidad clase de fragmento de la proteína N-terminal con sus 6 ABP 10. </li></ol> Tabla 1. Proteínas de la meta y los productos de la fusión con una etiqueta ABDz1 evaluado en un estudio de prueba de principio. Tres proteínas únicas humanos con distinto peso molecular, solubilidad y punto isoeléctrico fueron elegidos para la expresión y la purificación por el enfoque de afinidad ortogonal.

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

No hay conflictos de interés declarado.

El protocolo de purificación de afinidad ortogonal que aquí se presenta permite la purificación eficiente de una amplia gama de proteínas diana. Mediante la combinación de los inherentes al sitio de unión del dominio de unión a la albúmina, con una superficie de unión novela, una etiqueta pequeña proteína biespecíficos se desarrolló. Es muy sencillo de utilizar la etiqueta ya que simplemente puede ser clonado y expresado en la fusión a cualquier proteína de interés en un vector de expresión preferido. Equipamiento de serie disponible en la mayoría de los laboratorios pueden emplearse para la purificación de proteínas de dos pasos. Dado que la función de la etiqueta ABDz1 depende de plegamiento correcto del dominio para exponer de manera eficiente sus dos superficies de unión, el método se limita a las proteínas expresadas en forma soluble y no es adecuado para las purificaciones en condiciones desnaturalizantes. Los agentes reductores deben también ser evitados, ya que interfiere con la unión de ABDz1 a la Z-dominio en la matriz MabSelect Claro. Ortogonales purificación de afinidadción ofrece una alternativa útil a los métodos tradicionales con el fin de satisfacer las necesidades de proteínas de alta pureza en muchas aplicaciones exigentes. Al mismo tiempo, este método elimina la necesidad de etiquetar combinatoria cuando las diferentes estrategias de depuración se utilizan en la sucesión. Para aplicaciones que requieren una proteína diana nativo, un sitio de escisión de la proteasa pueden ser introducidos para hacer la eliminación enzimática de la etiqueta ABDz1 posible 11. Además, la etiqueta ABDz1 es el primer dominio biespecíficos pequeña proteína plegada y descritas hasta la fecha. Es único, ya que proporciona una estrategia de depuración que depende de dos interacciones de afinidad específicos. En el futuro sería interesante ampliar este concepto mediante el desarrollo de nuevas etiquetas biespecíficos que llevan a nuevas superficies de unión que son compatibles con las resinas de cromatografía fácilmente disponibles y baratos. Por ejemplo, mediante la sustitución de los aminoácidos involucrados en la unión a la albúmina con residuos básicos, una etiqueta compatible con ien la cromatografía de intercambio puede ser alcanzable. Un concepto similar se ha demostrado su eficacia por la ingeniería encargada de la Z-dominio para generar etiquetas conocido como Z 12 y Z ácido básico 13 compatible con el intercambio de aniones y cationes, respectivamente.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> E. coli RR1ΔM15 </td><td> American Type Culture Collection </td><td> 35102 </td></tr><tr><td> E. coli Rosetta (DE3) </td><td> Novagen </td><td> 70954 </td></tr><tr><td> Caldo de soja tríptico </td><td> Difco </td><td> 211822 </td></tr><tr><td> Extracto de levadura </td><td> Difco </td><td> 212720 </td></tr><tr><td> Vibra sonicador celular </td><td> Sonics y los materiales </td><td> - </td></tr><tr><td> HSA Sepharose </td><td> Pharmacia Biotech </td><td> Producto anterior </td></tr><tr><td> HiTrap Claro MabSelect </td><td> GE Healthcare </td><td> 11-0034-93 </td></tr><tr><td> HiTrap NHS-activado HP columna </td><td> GE Healthcare </td><td> 17-0716-01 </td></tr></tbody></table>

orthogonal protein purification facilitated by a small bispecific affinity tag

Debido a los altos costos asociados con la purificación de proteínas recombinantes de los protocolos deben ser racionalizadas. De alto rendimiento para los esfuerzos que hay una demanda de métodos generales que no requieren de la optimización de la proteína objetivo específico 1. Para lograr esto, las etiquetas de purificación que genéticamente puede ser fusionado con el gen de interés se utilizan 2. El asa de afinidad más utilizado es la etiqueta de hexa-histidina, que es adecuado para la purificación, tanto en condiciones nativas y desnaturalizantes 3. La carga metabólica para la producción de la etiqueta es baja, pero no proporciona la especificidad de hasta 1,2 competir cromatografía de afinidad estrategias basadas en. En este caso, una etiqueta de depuración biespecíficos con dos diferentes sitios de unión de un ácido amino 46, dominio de la proteína pequeña se ha desarrollado. El dominio de unión a la albúmina se deriva de la proteína G estreptocócica y tiene una fuerte afinidad inherente a la albúmina sérica humana(HSA). Once superficie expuesta aminoácidos, que no participan en la albúmina de unión 4, eran genéticamente al azar para producir una biblioteca combinatoria. La biblioteca de la proteína con la novela dispuestas al azar unión a la superficie (Figura 1) se expresó en las partículas de fago para facilitar la selección de carpetas de la tecnología de fagos. A través de varias rondas de biopanning contra un dímero Z-dominio derivado de la proteína A estafilocócica 5, una pequeña molécula, biespecíficos con afinidad por los HSA y el nuevo objetivo se identificó 6. El dominio de la nueva proteína, denominada ABDz1, se evaluó como una etiqueta de purificación de una selección de proteínas diana con distinto peso molecular, solubilidad y punto isoeléctrico. Tres proteínas diana se expresaron en Escherichia Escherishia con la etiqueta novela fusionada a su extremo N-terminal y posteriormente purificado por afinidad. Purificación inicial en una columna inmovilizada con HSA o Z de dominio dio lugar a relativamente los productos puros. Dos pasos de purificación de afinidad con la etiqueta biespecíficos produjo una mejora sustancial de la pureza de la proteína. Medios cromatográficos con la Z-dominio inmovilizadas, por ejemplo MabSelect Claro, están disponibles para la purificación de anticuerpos y HSA puede ser químicamente unida a los medios de comunicación para proporcionar la segunda matriz. Este método es especialmente ventajoso cuando hay una alta demanda en la pureza de la proteína diana recuperado. El bifunctionality de la etiqueta permite a dos pasos cromatográficos diferentes para ser utilizada en la carga metabólica en la expresión de acogida es limitada debido al pequeño tamaño de la etiqueta. Que proporciona una alternativa competitiva a los llamados de marcado combinatorio donde varias etiquetas se utilizan en combinación 1,7.

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

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Un novedoso y de alta eficiencia de dos pasos de cromatografía de afinidad protocolo ha sido desarrollado y es descrito en detalle. El método se basa en una etiqueta pequeña con dos purificación afinidades inherentes y es aplicable a una amplia gama de proteínas diana con diferentes propiedades.

Purificación de proteínas ortogonales Facilitado por un marcador de afinidad biespecíficos pequeños

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

17.0K vistas.  Royal Institute of Technology. Un novedoso y de alta eficiencia de dos pasos de cromatografía de afinidad protocolo ha sido desarrollado y es descrito en detalle. El método se basa en una etiqueta pequeña con dos purificación afinidades inherentes y es aplicable a una amplia gama de proteínas diana con diferentes propiedades.

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

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

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

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

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

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

El aislamiento de lábil Multi-complejos de proteínas por In vivo Control Celular entrecruzamiento y Inmuno-cromatografía de afinidad magnética

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

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

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 ...

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.

Identificación de los complejos de proteínas en<em&gt; Escherichia coli</em&gt; La purificación mediante afinidad del péptido secuencial en combinación con espectrometría de masas tándem

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

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimiento de purificación de afinidad de proteínas marcadas con recombinantes

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

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

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

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

GST-Su purificación: A dos pasos Affinity Protocolo Purificación Cediendo largos proteínas purificadas

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Purificación de proteínas libre de Método de Determinación afinidad de unión por termoforesis Microscale

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

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

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

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

Un Protocolo de fagos y de selección de afinidad utilizando cebos proteína recombinante

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

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

La identificación de la interacción proteína-proteína en<em&gt; Drosophila</em&gt; Jefes adultas por Tandem Affinity purificación (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Purificación de proteínas ortogonales Facilitado por un marcador de afinidad biespecíficos pequeños

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