Este trabajo fue apoyado por una subvención Wellcome Project (078043/Z/05/Z) para ROJW

PARTE A: alto rendimiento de crecimiento de cultivos bacterianos. 1. Las bacterias crecen Noche en 2 ml de medio de autoinducción Utilizando placas de 24 pocillos <ol><li> Esterilizar las placas de 24-así por microondas. </li><li> Inocular 1,5 ml de medio de autoinducción (durante la noche Medium Express) con recién crecido colonias bacterianas o stocks congelados glicerol. Normalmente inocular tres pocillos por mutante con tres colonias individuales clonados. Reserva seis pozos para los controles positivos y negativos. Para los controles positivos que crecen tres clones de tipo silvestre y para los controles negativos que crecen 2 clones que han sido transformadas con un plásmido que no expresa. Entrada para la esterilización suficiente de la placa, dejando así espacio en blanco uno para el control del medio de sólo. </li><li> Cultivar las células durante 18 horas a 37 ° C y agitación a 250 rpm. Usamos lac inducible BL21 (DE3) Rosetta 2 células. Medio de autoinducción contiene una mezcla de glucosa y lactosa. Las bacterias inicialmentealimentan de glucosa y luego comenzar a utilizar la lactosa, que también induce la expresión de las proteínas recombinantes. </li><li> Retire la tapa y coloque la placa en la plataforma robótica. </li></ol> PARTE B: Robótica purificación de proteínas recombinantes. 2. Prepare la plataforma robótica <ol><li> Completar hasta el tampón de lavado que consiste en 20 mM de imidazol, 0,1% Triton X-100, 0,5 M NaCl, 20 mM Tris-acetato, pH 7,9, 10 mM de MgOAc 2, 0,7 mM ZnOAc 2, 10% de glicerol, y el tampón de elución consistente de 0,5 M imidazol, 0,1% Triton X-100, 0,5 M NaCl, 20 mM Tris-acetato, pH 7,9, 10 mM de MgOAc 2, 0,7 mM ZnOAc 2, 10% de glicerol. Estos tampones se utilizan para lavar las perlas después de las proteínas etiquetadas se han unido a ellos, o para eluir las proteínas de las perlas, respectivamente. </li><li> Completar el diluyente bacteriana (100 ml de agua destilada con 15 l de reactivo antiespumante). Esta solución será utilizadapara hacer diluciones de los cultivos bacterianos durante la noche para la densidad óptica (A600) mediciones. La presencia de agente antiespumante en el diluyente evita la formación de burbujas de aire que podrían interferir con las mediciones lector de placas. </li><li> Hacer la solución de lisis reactivo que consiste en FastBreak 10x, 2 lysonase l por muestra y 15 mM MgOAc 2. FastBreak contiene una mezcla de detergentes y sales que se rompen las paredes celulares de las bacterias y facilitar la liberación de las proteínas intracelulares. Lysonase es una mezcla patentada de lisozima y una nucleasa. La lisozima asiste en la interrupción de la pared celular y la nucleasa digiere las Autorizaciones de ácidos nucleicos bacterianos. </li><li> Opcional: Haga una solución 6 M de clorhidrato de guanidina. Algunas proteínas recombinantes tienden a adherirse a las agujas recubiertos de teflón de pipeteado. La guanidina clorhidrato desnaturaliza las proteínas de la solución y los lava fuera más efectiva que el agua que se usa rutinariamente para enjuagar las puntas lavables robóticas de pipeteo después de cada step. </li><li> Preparar la BCA (ácido bicinconínico) de reactivo cuantificación de proteínas por la solución de mezcla de ácido bicinconínico (reactivo A) y la solución de sulfato de cobre (reactivo B): Los enlaces peptídicos reducir Cu 2 + de la CuSO 4 componente presente en el reactivo BCA a Cu 1 + mediante el cual la cantidad de Cu 1 + es proporcional al número de enlaces peptídicos presentes en la solución. En una segunda etapa, dos moléculas de ácido bicinconínico quelato de Cu 1 + que resulta en un cambio de absorbancia a 562 nm, lo que resulta en un color púrpura. La reacción de color es dependiente del tiempo y por lo general necesita varias horas para ser definitivo. Después de que el color es estable durante varias horas. </li><li> Llene cubetas o placas del tamaño adecuado y colocarlos en su posición pre-asignados en la plataforma robótica. </li><li> Coloque un 24-así placa para los residuos hidrocloruro de guanidina, dos placas transparentes de 96 pocillos para OD 600 y medidas de absorbancia y uno azul placa de 96 pocillos para the purificado proteínas en sus posiciones sobre la plataforma. Coloque una placa de 96 pozos profundos en el soporte magnético. </li><li> Diluir MagneHis Ni-partículas que se unen a las proteínas mediante la formación de quelatos con sus His-tag de 5 veces en agua destilada y llenar ellos en la unidad de talón-agitación. Encienda el agitador para mantener las cuentas en suspensión. </li><li> Encienda la plataforma robótica y lave las agujas de pipeteo durante varios minutos para eliminar las burbujas de aire que de otra forma interferir con la precisión de pipeteo. Las agujas de pipeteo reutilizables se lavan entre los pasos de pipeteo individuales. Alternativamente, puntas desechables podrían ser utilizados. Todas las etapas subsiguientes se llevan a cabo robóticamente. El protocolo de robótica está disponible bajo petición. </li></ol> 3. El crecimiento celular se comprobarse midiendo la DO600 <ol><li> 10 l de cultivo de una noche se diluye en 90 l de solución diluyente. </li><li> Medir el diámetro exterior 600 para asegurar que las bacterias han crecido hasta densidades similares(Figura 1). </li></ol> 4. Las células se dividen para liberar las proteínas y permitir a bolas de unión <ol><li> 100 l de suspensión magnética Ni-talón se distribuyen en cada pocillo de una segunda placa de 24 pocillos. </li><li> 900 l de cada cultivo bacteriano a pequeña escala se transfiere a la placa de 24-pocillos que contiene las perlas. Añadir 100 l de 10 veces el FastBreak / lysonase mezcla. </li><li> La placa de 24-así se transfiere a la plataforma de agitación para una agitación rápida (800 rpm) durante 30 min a temperatura ambiente. Las paredes celulares bacterianas se rompen mediante una combinación de fuerzas mecánicas y reacciones químicas y las proteínas producidas por recombinación se liberan en solución. Con sus etiquetas de afinidad que de inmediato se unen las perlas paramagnéticas quelantes de níquel. </li></ol> 5. Las perlas se lavan <ol><li> Los celulares lisados ​​que contienen las perlas magnéticas resuspendidas se transfirieron a una placa de 96 pozos profundos que se coloca en un soporte con magnetic varillas que se deslizan entre los pocillos. Las placas de 96 pocillos tienen un cuadrado en forma de cono inferior que permite una fácil eliminación del sobrenadante. Los pozos pueden contener volúmenes de hasta 2,1 ml, pero se llenan con volúmenes mucho más pequeños. Esto nos permite realizar vigorosos pasos que sacuden sin muestra la contaminación cruzada por salpicaduras. </li><li> Las puntas de pipeta se lavan entre cada uno de los pasos de pipeteado con 6 M guanidina-HCl. Este paso es crucial para la purificación de TFIIB y otras proteínas &quot;pegajosas&quot; para evitar la contaminación cruzada. Con otras proteínas, puede ser suficiente para enjuagar las agujas extensamente con agua entre las etapas de pipeteado. </li><li> Las barras magnéticas del soporte magnético atraer las perlas paramagnéticas, tirando de ellos lejos de los centros de los pocillos y se permiten las agujas de pipeteado libre acceso al sobrenadante. El sobrenadante se desecha. </li><li> 500 l de tampón de lavado se añade a la primera placa de 24 pocillos y posteriormente transferido a la placa de 96 pocillos a eNsure la transferencia completa de las cuentas. La placa de 24-así se elimina de la coctelera. </li><li> Otro l de tampón de lavado 500 se añade directamente a la placa de 96-pocillos, la placa se transfiere al agitador y se agita vigorosamente durante 1 min. La placa se mueve hacia atrás en el soporte magnético y se desechó el tampón de lavado. </li><li> Este paso se repitió dos veces y el procedimiento de lavado ha terminado por la eliminación de restos de amortiguamiento de la placa. </li></ol> 6. Eluir las proteínas en tampón de elución <ol><li> 100 l de tampón de elución se añadió a las perlas, la placa se mueve al agitador y se agita vigorosamente durante 30 min a temperatura ambiente. </li><li> La placa se mueve de nuevo al agitador y el eluato, que contiene la proteína recombinante purificada, se transfirieron a una placa nueva. </li></ol> 7. Medir las concentraciones de las proteínas purificadas <ol><li> 190 l de mezcla de reactivo de BCA se transfiere a un claro placa de 96 pocillos y 10 l de la protein solución añadida. </li><li> Después de varias horas de incubación (típicamente 5-6 o durante la noche), se mide la absorbancia y compararlo con estándares BSA para determinar las concentraciones de cada una de las preparaciones de proteínas purificadas (Figura 2). </li><li> Las proteínas purificadas se pueden utilizar ahora para aplicaciones posteriores a las concentraciones correspondientes (figura 3). </li></ol> 8. Los resultados representativos El protocolo de purificación ofrece dos etapas de control de calidad, ejemplos de los cuales se muestran. Que son capaces de identificar y documentar los problemas potenciales en la etapa de crecimiento bacteriano (Figura 1) y más tarde después de evaluar los rendimientos de las proteínas purificadas (Figura 2). Por lo general purificar proteínas y prueba de ellos por triplicado. Esto, en combinación con los dos pasos de control de calidad, nos da confianza de que cualquier variación se observó en los ensayos funcionales se deben a la phenotyp mutantee (Figura 3) y no causado por las variaciones experimentales o purificaciones fallidos. Los rendimientos obtenidos típicamente en el intervalo desde 50 hasta 200 g y son más que suficientes para diversos ensayos funcionales. <img alt="Figura 1" src="/files/ftp_upload/4110/4110fig1.jpg" /> Figura 1. Histograma de las mediciones de densidad óptica de una placa de 24 pocillos con cultivos de una noche. Tres clones de seis variantes TFIIB mutantes así como de la TFIIB tipo salvaje se han cultivado. Dos clones que llevan un plásmido que no expresa bien y con un medio de sólo sirven como controles negativos. Las mediciones de OD muestran que existen pequeñas variaciones en las tasas de crecimiento entre las distintas culturas. <img alt="Figura 2" src="/files/ftp_upload/4110/4110fig2.jpg" /> Figura 2. Rendimientos de proteína obtenidos a partir de estos cultivos como se determina mediante un ensayo BCA y se confirmó por SDS PAGE. Una de las variantes no se expresa en niveles altos. En comcombinación con la figura 1, se puede concluir que esto no era debido al crecimiento celular diferencial, pero debido a la expresión de la proteína no siendo inducido correctamente. <img alt="Figura 3" src="/files/ftp_upload/4110/4110fig3.jpg" /> Figura 3. Representante resultado de un ensayo de transcripción. Se midió la actividad de estimulación de variantes TFIIB en la producción de pequeños transcritos abortivos por RNAP. Aquí, los efectos de la estimulación de una biblioteca completa de sustituciones de aminoácidos del residuo TFIIB K87 se muestran. El alto grado de reproducibilidad es confirmado por las tasas de error pequeños. Una muestra de gel que muestra el rendimiento de los tres mutantes en comparación con el tipo salvaje (WT), controles negativos (NC) y tampón de elución sólo controla se representa debajo.

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No hay conflictos de interés declarado.

El método automatizado de purificación de la proteína recombinante descrita aquí permite la producción y purificación de un gran número de proteínas mutantes en un formato de pequeña escala bajo condiciones altamente reproducibles con una mínima intervención humana. Figuras 1 y 2 muestran los resultados de los controles sistemáticos de calidad y ejemplos de la proteínas purificadas. Figura 3 muestra que los factores de transcripción purificados usados ​​en este ejemplo realizar de una manera altamente reproducible en ensayos funcionales. A pesar de que el procedimiento fue desarrollado para la purificación de archaeal TFIIB, es ampliamente aplicable para la purificación de proteínas etiquetadas por afinidad. El uso de tales protocolos de purificación automatizados por lo tanto será significativamente facilitar el análisis bioquímico de proteínas recombinantes y por lo tanto mejorar nuestra comprensión de las interacciones proteína-proteína en una escala que es difícil de conseguir manualmente.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Overnight Express Media TB instantánea </td><td> Merck Chemicals Ltd </td><td> 71491-4 </td><td></td></tr><tr><td> FastBreak </td><td> Promega Ltd. </td><td> V8573 </td><td></td></tr><tr><td> Lysonase Reactivo bioprocesamiento / ml 1 </td><td> Merck Chemicals Ltd </td><td> 71230 </td><td></td></tr><tr><td> Antiespumante 204 </td><td> Sigma-Aldrich Company Ltd </td><td> A6426 </td><td></td></tr><tr><td> MagneHis Ni-Partículas </td><td> Promega </td><td> V8565 </td><td></td></tr><tr><td> Imidazol </td><td> Sigma-Aldrich Company Ltd </td><td> 56750 </td><td></td></tr><tr><td> Trizma base </td><td> Sigma-Aldrich Compcualquier Ltd. </td><td> 93362 </td><td></td></tr><tr><td> NaCl </td><td> VWR </td><td> 27810.295 </td><td></td></tr><tr><td> Ácido bicinconínico determinación de proteínas </td><td> Sigma-Aldrich Company Ltd </td><td> BCA1-1KT </td><td></td></tr><tr><td> Deep Well Plate 2,2 ml Square Wells PP 10 UDS </td><td> Anachem Ltd </td><td> 1810-00 </td><td></td></tr><tr><td> Microplaca 96 MicroWell poliestireno de fondo plano de 12 mangas no se trata de 5 unidades cada claro 0,4 ml de volumen y 128 mm x 86 mm Thermo Scientific Nunc </td><td> Fisher Scientific Ltd. </td><td> DIS-984-090m </td><td></td></tr><tr><td> Microplaca Azul </td><td> VWR </td><td> NUNC367001 </td><td></td></tr><tr><td> 24-Bueno Bloques RB (24) </td><td> Qiagen </td><td> 19583 </td><td></td></tr><tr><td> Hidrocloruro de guanidina </td><td> VWR </td><td> ALFAA13543.0B </td><td></td></tr><tr><td> Aguja lavable para TheOnyx </td><td> Aviso GmbH </td><td> 8152-317001 </td><td></td></tr><tr><td> Reactivo Rack para perlas magnéticas </td><td> Aviso GmbH </td><td> 8152-035003 </td><td></td></tr><tr><td> Lector de Placas Synergy HT </td><td> BioTek </td><td> 4200-000043 </td><td></td></tr><tr><td> Robotic TheOnyx Plataforma 44OH/150/100 </td><td> Aviso GmbH </td><td> 8145-050046 </td><td></td></tr><tr><td> Agitador de microplacas Variomag Teleshaker </td><td> Inheco </td><td> 3800047 </td><td></td></tr><tr><td> De 96 pocillos Tipo de imán A </td><td> Qiagen </td><td> 36915 </td><td></td></tr></tbody></table>

high-throughput purification of affinity-tagged recombinant proteins

Cristalografía de rayos X es el método de elección para obtener una vista detallada de la estructura de las proteínas. Tales estudios deben ser complementados por otros análisis bioquímicos para obtener una detallada comprensión de las relaciones estructura / función. Los avances en la tecnología de síntesis de oligonucleótidos y el gen de hacer grandes estrategias de mutagénesis cada vez más factibles, incluyendo la sustitución de residuos diana por todos los 19 aminoácidos distintos. Ganancia o pérdida de función de los fenotipos después permitir conclusiones sistemáticas a extraer, tales como la contribución de los residuos particulares para la estabilidad de la proteína actividad catalítica, y / o proteína-proteína interacción especificidad. Con el fin de atribuir a los diferentes fenotipos de la naturaleza de la mutación - en lugar de a la fluctuación de las condiciones experimentales - es vital para purificar y analizar las proteínas de una manera controlada y reproducible. De alto rendimiento estrategias y la automatización de los protocolos de manuales sobrerobotizados de manipulación de líquidos plataformas han creado oportunidades para llevar a cabo tales procedimientos complejos moleculares biológicos con poca intervención humana y las tasas mínimas de error 1-5. Aquí, se presenta un método general para la purificación de Su-etiquetados proteínas recombinantes en una forma de alto rendimiento. En un estudio reciente, se aplicó este método a una detallada estructura-función de investigación de TFIIB, un componente de la maquinaria de transcripción basal. TFIIB es indispensable para el promotor dirigido por la transcripción in vitro y es esencial para el reclutamiento de la polimerasa de ARN en un complejo de preiniciación 6-8. TFIIB contiene un dominio de unión flexible que penetra en la hendidura del sitio activo de la ARN polimerasa 9-11. Este dominio de unión confiere dos actividades cuantificables bioquímicamente en TFIIB, a saber, (i) la estimulación de la actividad catalítica durante la &#39;abortivo&#39; fase de iniciación de transcripción, y (ii) una contribución adicional a lareclutamiento específico de la ARN polimerasa en el complejo de preiniciación 4,5,12. Hemos explotado el método de purificación de alto rendimiento para generar sustitución de un solo, doble y triple y mutaciones supresiones dentro del enlazador TFIIB y para posteriormente analizar en ensayos funcionales para su efecto de estimulación de la actividad catalítica de la polimerasa de ARN 4. En total, hemos generado, se purificó y se analizó 381 mutantes - una tarea que habría sido mucho tiempo y es laborioso de realizar manualmente. Hemos producido y se analizaron las proteínas en multiplicates lo que nos permitió apreciar las variaciones experimentales y nos dio una idea clara de la reproducibilidad de los resultados. Este método sirve como un protocolo genérico para la purificación de Su-etiquetados proteínas y se ha utilizado con éxito para purificar otras proteínas recombinantes. En la actualidad está optimizado para la purificación de proteínas de 24, pero se puede adaptar a purificar hasta 96 proteínas.

Watch this Scientific Journal Video about High-throughput Purification of Affinity-tagged Recombinant Proteins at JoVE.com

High-throughput Purification of Affinity-tagged Recombinant Proteins

Se describe un método para la purificación por afinidad de etiquetado de proteínas recombinantes utilizando robótica de manejo de líquidos. Este método es generalmente aplicable a la purificación a pequeña escala de solubles Su-etiquetados proteínas en un formato de alto rendimiento.

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 ...

high-throughput-purification-of-affinity-tagged-recombinant-proteins

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JoVE Journal

Biology

14.1K vistas.  Imperial College London. Se describe un método para la purificación por afinidad de etiquetado de proteínas recombinantes utilizando robótica de manejo de líquidos. Este método es generalmente aplicable a la purificación a pequeña escala de solubles Su-etiquetados proteínas en un formato de alto rendimiento.

Video: High-throughput Purification of Affinity-tagged Recombinant Proteins

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

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Expresión de proteínas recombinantes en la levadura metilotrófica Pichia pastoris</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

La levadura de alto rendimiento de la pantalla sobreexpresión plásmido

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

El uso de un robot para la cristalización de alto rendimiento de proteínas de membrana en mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

High Throughput microinyecciones de Sea Urchin cigotos

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Producción eficiente y purificación de recombinante murino kindlin-3 a partir de células de insecto para estudios biofísicos

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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

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

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

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

High Throughput Screening Expresión cuantitativa y Purificación Aplicada a Recombinante Disulfide ricos en proteínas del veneno Producido en<em&gt; E. coli</em

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

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

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

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

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

La purificación no cromatográfico de polipéptidos recombinantes Elastina-como y sus fusiones con Péptidos y Proteínas de<em&gt; Escherichia coli</em

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

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

De Alto Rendimiento La titulación de luciferasa que expresan virus recombinantes

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...