Los autores desean dar las gracias al Sr. Luis F. Delgadillo (Departamento de Ingeniería Química y Biomolecular, Universidad de Ohio), al Sr. Matthew H. Williams (Departamento de Ingeniería Química y Biomolecular, Universidad de Ohio) y al Dr. Filiberto Cedeno-Laurent (Brigham and Women's Hospital y Harvard Medical School) por su asistencia técnica experta. Los autores también desean agradecer a los estudiantes de invierno de 2011 CHE 404/BME 504 y otoño de 2012 CHE 4830/BME 5830 (Departamento de Ingeniería Química y Biomolecular y Programa de Ingeniería Biomédica, Universidad de Ohio) por su asesoramiento técnico y discusión. Este trabajo fue apoyado por la Fundación Nacional de Ciencias (NSF) Mayor Instrumentación de Investigación CBET-1039869 (MMB), NSF CBET-1106118 (MMB), Dermatology Foundation Research Grant A050422 (SRB), Institutos Nacionales de Salud (NIH) NCI subvención 1R15CA161830-01 (MMB), NIH Kirschstein-NRSA Beca Postdoctoral F32CA144219-01A1 (SRB), NIH / NCI R01CA173610 (CJD), y NIH NCCAM subvención R01AT004628 (CJD).

1. Verificar la afinidad por la proteína A
<ol><li>Verificar que la proteína que expresa Fc (proteína de fusión recombinante o anticuerpo IgG, hasta ahora denominada "proteína") tiene una afinidad aceptable por la proteína A14-17 antes de usar el adsorbente de membrana de la proteína A, y probar la presencia de la proteína en la solución madre (por ejemplo, sobrenadante de cultivo celular clarificado por centrifugación a 1.000 x g durante 5 min a células suspendidas de pellets). Utilice citometría de flujo, Western blotting, ELISA, etc. para detectar la función de la proteína y/o la presencia de Fc, en función de la preferencia del laboratorio. Idealmente, cuantificar la proteína en el sobrenadante de cultivo celular (paso 6) para evitar exceder la capacidad del adsorbente de membrana de la proteína A. Proceda si se detecta la proteína.</li>
</ol>2. Preparar la proteína A adsorbente de membrana y sobrenadante de cultivo celular
<ol><li>Siga las instrucciones del fabricante para preparar el adsorbente de membrana. Nunca deje que el aire entre en el adsorbente de membrana. Todas las soluciones perfundidas a través del adsorbente de membrana deben estar a temperatura ambiente y prefiltradas utilizando un filtro de 0,22 μm. Llene una jeringa de 10 ml con solución salina tamponada de fosfato (DPBS) de Dulbecco filtrada por 0,22 μm o tampón de burbujas de aire de elección y descarga. Perfundir DPBS para eliminar la solución de almacenamiento y equilibrar el adsorbente de membrana inmediatamente antes de su uso.</li>
	<li>Filtre el sobrenadante de cultivo celular inmediatamente antes de la perfusión a través del adsorbente de membrana, utilizando una unidad de filtración estéril de 0,22 μm impulsada por vacío.</li>
</ol>3. Cargue el adsorbente de membrana de la proteína A
<ol><li>Aspirar el sobrenadante de cultivo celular en una jeringa Luer Lock (30 ml o más) y descargar cualquier burbuja de aire.</li>
	<li>Montar materiales y equipos como se muestra en la Figura 1. Conecte la jeringa a la entrada del adsorbente de membrana. Conecte un tubo flexible a la salida del adsorbente de membrana. Si lo desea, coloque un filtro de jeringa de 0,22 μm entre la jeringa y el adsorbente de membrana. Use un matraz o botella para atrapar el flujo (también conocido como filtrado), del adsorbente.</li>
	<li>Ajuste la bomba de la jeringa a la velocidad de flujo volumétrica deseada, pero no exceda la tasa de flujo recomendada por el fabricante(por ejemplo, 10 ml / min). Perfundir el sobrenadante de cultivo celular a través del adsorbente de membrana, recogiendo el flujo a través de un castor u otro recipiente. Vuelva a cargar la jeringa con los sobrenadantes necesarios.</li>
	<li>Si lo desea, pruebe el flujo a través de la presencia de proteína utilizando citometría de flujo, Western blotting, ELISA, etc. basado en la preferencia del laboratorio. Por lo general, no es necesario volver a dispersar el flujo a través de.</li>
</ol>4. Proteína Elute del adsorbente de membrana
<ol><li>Lave el adsorbente de membrana con 10 ml de DPBS para eliminar cualquier proteína no ligada.</li>
	<li>Proteína elute del adsorbente de membrana al caudal deseado(por ejemplo, 1 ml/min) utilizando 10-15 ml de tampón de elución(por ejemplo, tampón de elución a base de aminas (pH 2.8)). Atrapar eluido en un tubo que contenga tampón neutralizante(por ejemplo, 1 M Tris (pH 9,4)) al 10% del volumen de elución (1,0-1,5 ml).</li>
	<li>Alternativamente, la proteína elute en un ml se incrementa en tubos que contienen 100 μl de tampón de neutralización, utilizando un volumen total de 10-15 ml de tampón de elución. Utilice el método de caracterización preferido para probar cada fracción para la presencia de proteína.</li>
	<li>Regenerar el adsorbente de membrana de acuerdo con las instrucciones del fabricante. Perfundir 10 ml de DPBS filtrados a 0,22 μm o tampón de su elección, luego 10 ml de NaOH de 0,22 μm filtrados a 50 mM en 1 N NaCl, y finalmente 10 ml de DPBS filtrados a 0,22 μm. Llene el adsorbente de membrana con etanol al 20% en DPBS para su almacenamiento a largo plazo a 4 ºC.</li>
</ol>5. Concéntrese y descongele la proteína
<ol><li>Depositar todos los eluos (o fracciones de elución que contengan proteínas según lo determinado en el paso 4.3) en una unidad de filtro centrífugo de corte de peso molecular de 10 kDa. Siga las instrucciones del fabricante para la centrifugación.</li>
	<li>Dializar el retentado en una unidad de diálisis de pequeño volumen (corte de peso molecular de 10 kDa) contra el tampón de elección, siguiendo las instrucciones del fabricante. Es posible que sea necesario agregar un tampón al material dializado si la precipitación de proteínas es una preocupación. Si se desea, el material dializado se puede refrigerar hasta que se pueda realizar el paso 6, pero no se recomienda el almacenamiento a largo plazo sin conservantes.</li>
</ol>6. Cuantificar y caracterizar el producto purificado en un procedimiento de western blotting acelerado
<ol><li>Resolver los estándares de proteínas purificadas y Fc en el gel de elección por SDS-PAGE, en condiciones reductoras o no reductoras como se desee18-20. Utilice un mini gel en lugar de gel midi para minimizar el tiempo de ejecución.<ol>
<li>Si aún no se conoce, determine el rango de estándares Fc en qué banda la señal varía linealmente con respecto a la cantidad de carga(por ejemplo, 0,1-1 mg). Use el mismo gel, condiciones de funcionamiento, condiciones de transferencia y reactivos que se utilizarán para cuantificar y caracterizar la proteína purificada.</li>
			<li>Cargue múltiples cantidades de muestras de proteína purificada, incluidas las muestras diluidas con DPBS, para asegurarse de que las señales de banda de proteína purificada estén dentro del rango de señal lineal de los estándares Fc en el análisis de imágenes.</li>
		</ol>
</li>
	<li>Transfiera proteínas del gel a una membrana de fluoruro de polivinidideno (PVDF) en un sistema tampón discontinuo utilizando un blottersemidry 13,siguiendo las instrucciones del fabricante del blotter. El tiempo de transferencia es típicamente de 5-10 min. Si se desea, Coomassie manchar el gel después de la transferencia para verificar la eficiencia de la transferencia21.</li>
	<li>Realizar inmunoblotting con anti-Fc o anti-IgG conjugado con fosfatasa alcalina (AP) o peroxidasa de rábano de caballo (HRP), basado en la preferencia de laboratorio, utilizando un sistema de detección de proteínas asistidas por vacío. El tiempo de immunoblotting es típicamente menos de 1 hora.</li>
	<li>Desarrollar immunoblot utilizando el sustrato y el método de elección(por ejemplo, sustrato AP y quimioluminiscencia mejorada).</li>
	<li>Cuantifique la proteína purificada por análisis de imagen. Realice una regresión lineal en las señales de banda de los estándares Fc. SiR2>0,90, utilice la ecuación de la línea para calcular la cantidad de proteínas a partir de sus señales de banda. Tenga en cuenta la dilución de la muestra si es necesario. Dado que la cuantificación de la proteína se realiza sobre la base de Fc, ajuste el valor para las diferencias de peso molecular entre la proteína y Fc. SiR2<0,90, repita el paso 6 en su totalidad.</li>
	<li>Validar la proteína utilizando ensayos funcionales o métodos para detectar Fc a través de citometría de flujo, Western blotting, ELISA, etc. basado en la preferencia del laboratorio.</li>
	<li>Diluya la proteína a la concentración deseada y/o agregue los conservantes o estabilizadores deseados a la solución de proteína purificada antes de alícuota para su almacenamiento a largo plazo, congelado o de otro tipo.</li>
</ol>

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No hay nada que revelar.

El protocolo descrito en este documento fue desarrollado para aislar y caracterizar rápidamente una proteína quimérica de fusión que expresa fc humano (Gal-1hFc), sin comprometer significativamente la recuperación del producto o inflar el costo. Los componentes clave del flujo de trabajo optimizado son un adsorbente de membrana de proteína A para el aislamiento, y la transferencia de semisel y el inmunoblotting asistido por vacío para la caracterización por western blotting.
La dismin...

El aislamiento de anticuerpos y proteínas recombinantes que expresan fragmentos de inmunoglobulina G (IgG) Fc de sobrenadantes de cultivos celulares y soluciones celulares diluidas se puede lograr utilizando cromatografía de afinidad de proteínas A. En los laboratorios de ciencia básica e ingeniería y en la industria, las columnas empaquetadas con agarosa recubierta de proteína A porosa, vidrio o perlas poliméricas se utilizan más comúnmente para la cromatografía de afinidad, a pesar de los altos costos financieros y los largos tiempos de procesamiento1-3. Es muy apreciado que la cromatografía de afinidad representa el mayor gasto y cuello de botella de procesamiento tanto en entornos de laboratorio como industriales2,4. Como resultado, se han desarrollado numerosas mejoras para disminuir el costo y el tiempo de procesamiento sin sacrificar la recuperación general del producto del tren de proceso1,2,5-7. Particularmente prometedor para el aislamiento de anticuerpos y otras proteínas que expresan Fc es la cromatografía de afinidad utilizando un adsorbente de membrana de proteína A2,5,7-10. Mientras que la captura de Fc en cromatografía de columna está limitada por difusión (esdecir, la proteína que expresa Fc debe difundirse dentro y a través de los poros internos para alcanzar la mayoría de la proteína A) con alta caída de presión a través de la columna, el transporte de masa en los adsorbers de membrana es impulsado por la convección a granel, lo que resulta en la evitación de las limitaciones de difusión8,9,11,12. Además, la caída de presión a través del adsorbente de membrana es baja, lo que permite tasas de flujo de perfusión más rápidas en comparación con las columnas empaquetadas con perlas8,9,11,12. Por lo tanto, para el aislamiento a escala de laboratorio, se predice que la cromatografía de membrana reducirá el tiempo de aislamiento en varias horas y aumentará la captura del producto en comparación con la cromatografía de columna. Además, se han propuesto modelos matemáticos para la adsorción de IgG en adsorbentes de membrana8,9,11,12,permitiendo así a los usuarios finales predecir el rendimiento en el laboratorio.
Otro cuello de botella en los laboratorios de ciencia básica e ingeniería es la caracterización de la proteína que expresa Fc. Sin embargo, la selección de métodos apropiados para completar los ensayos de caracterización, como las manchas occidentales, puede reducir en gran medida el tiempo dedicado a las pruebas de productos. Por ejemplo, la transferencia de semisido en un sistema tampón discontinuo puede lograr la transferencia electroforética de proteínas de un gel SDS-PAGE a una membrana de polivinilo difluoridina (PVDF) en cuestión de minutos, en lugar de 1-2 horas en la transferencia del tanque (húmedo) en un sistema tampón continuo13.
Un protocolo rápido, barato, y eficaz para aislar y para caracterizar una proteína quimérica de la fusión que expresa un fragmento del Fc de IgG humano se describe adjunto. La mayoría de los equipos están fácilmente disponibles en los laboratorios estándar de ciencias biomédicas básicas, biología, química y (bio)ingeniería química, y todos los reactivos están disponibles comercialmente. Aunque se generaron resultados representativos a partir del aislamiento y caracterización de una proteína de fusión quimérica Murina Galectina-1/Humana Fc (Gal-1hFc), el protocolo simplificado se puede aplicar al aislamiento de otras moléculas que poseen un fragmento Fc, como anticuerpos, fragmentos Fc ellos mismos, u otras proteínas de fusión Fc. Nuestras mejoras disminuyeron drásticamente el tiempo de procesamiento (tan solo 4 horas, pero más típicamente ~ 9 horas, en comparación con más de 20 horas de trabajo) mientras lograba una recuperación del producto similar o mejorada en comparación con los métodos estándar.

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			<td height="42" style="height:42px;width:313px;">Protein A membrane adsorber (Sartobind Protein A 2 ml)&#160;</td>
			<td style="width:191px;">Sartorius-Stedim</td>
			<td style="width:147px;">93PRAP06HB-12--A</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Amicon Ultra centrifugal filter, 10 kDa, 4 ml</td>
			<td style="width:191px;">Millipore</td>
			<td>UFC801008</td>
			<td>Use 15 ml volume if needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sterile vacuum filter unit, 0.22 &#181;m, 500 ml</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">10 ml Syringes with Luer Lock tips</td>
			<td>BD</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">30 ml Syringes with&#160; Luer Lock tips</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tygon lab tubing (1/16 in x 1/8 in x 50 ft)</td>
			<td>Cole Parmer</td>
			<td>WU-95903-16</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)</td>
			<td>Thermo Scientific</td>
			<td>69576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. antibody collection tray</td>
			<td>EMD Millipore</td>
			<td>WBAVDABTR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. single well blot holder</td>
			<td>EMD Millipore</td>
			<td>WBAVDBH01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Elution buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tris, ultra pure</td>
			<td>Fisher Scientific</td>
			<td>819623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">DPBS</td>
			<td>Thermo Scientific&#160;</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">DPBS with Ca2+/Mg2+</td>
			<td>Life Technologies</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">BSA</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Human Fc</td>
			<td>Bethyl Research Laboratories</td>
			<td>&#160;P80-104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-APC</td>
			<td>Jackson Immunoresearch</td>
			<td>109136170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-AP</td>
			<td>Bio-Rad</td>
			<td>170-5018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Alkaline phosphatase substrate</td>
			<td>Promega</td>
			<td style="width:147px;">S3841</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Flow cytometry tubes (5 ml polystyrene or polypropylene)</td>
			<td>BD</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;"></td>
			<td style="width:191px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>55-2226</td>
			<td style="width:173px;">A peristaltic pump is also acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Protein gel electrophoresis system&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>552BR094876</td>
			<td style="width:173px;">Any gel electrophoresis system is acceptable, but mini gel systems run fastest</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Semidry blotter&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td style="width:147px;">690BR006163</td>
			<td style="width:173px;">Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:313px;">SNAP i.d. vacuum-assisted protein detection system</td>
			<td style="width:191px;">EMD Millipore</td>
			<td>WBAVDBASE</td>
			<td style="width:173px;">An upgraded model has replaced this specific instrument, but either should work just as well</td>
		</tr>
	</tbody>
</table>

isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow

La purificación a escala de laboratorio a escala industrial de biomoléculas de sobrenadantes de cultivos celulares y soluciones celulares lisadas se puede lograr mediante cromatografía de afinidad. Mientras que la cromatografía de afinidad utilizando perlas de agarosa de proteína A porosa empaquetadas en columnas es posiblemente el método más común de aislamiento a escala de laboratorio de anticuerpos y proteínas recombinantes que expresan fragmentos de Fc de IgG, puede ser un proceso lento y costoso. Las limitaciones de tiempo y financieras son especialmente desalentadoras en pequeños laboratorios de ciencias básicas que deben recuperar cientos de microgramos a cantidades de miligramos de proteína de soluciones diluidas, pero carecen de acceso a sistemas de administración de líquidos de alta presión y / o personal con experiencia en bioseparaciones. Además, la cuantificación y caracterización del producto también puede alargar excesivamente el tiempo de procesamiento durante varios días de trabajo e inflar los gastos (consumibles, salarios, etc.). Por lo tanto, se necesita un protocolo rápido, económico pero efectivo para el aislamiento a escala de laboratorio y la caracterización de anticuerpos y otras proteínas que poseen un fragmento de Fc. Con este fin, hemos ideado un protocolo que puede ser completado por personal técnico de experiencia limitada en menos de 9 horas (aproximadamente un día de trabajo) y tan rápido como 4 horas, en comparación con los métodos tradicionales que exigen más de 20 horas de trabajo. La mayoría del equipo requerido está fácilmente disponible en los laboratorios estándar de ciencia biomédica, bioquímica e ingeniería (bio)química, y todos los reactivos están disponibles comercialmente. Para demostrar este protocolo, los resultados representativos se presentan en los cuales el galectin-1 murine quimérico fundido al Fc humano (Gal-1hFc) del sobrenadante del cultivo celular fue aislado usando un adsorber de la membrana de la proteína A. Gal-1hFc purificado fue cuantificado usando un procedimiento occidental apresurado del análisis que borraba y caracterizado usando cytometry de flujo. El flujo de trabajo optimizado puede modificarse para otras proteínas que expresan Fc, como los anticuerpos, y/o alterarse para incorporar métodos alternativos de cuantificación y caracterización.

El protocolo simplificado para el aislamiento y la caracterización de las proteínas que expresan Fc se utiliza rutinariamente para procesar la galectina murina quimérica-1 fusionada a Fc humano (Gal-1hFc) a partir de sobrenadantes de cultivo celular diluidos. El diagrama de flujo de la figura 2 ilustra el flujo de trabajo y la hora de cada paso del protocolo. Para un lote típico de 300 ml de sobrenadante, el tiempo total de procesamiento es de aproximadamente 9 horas cuando se realiza la prueba opcio...

Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

En comparación con la cromatografía de afinidad tradicional utilizando columnas empaquetadas de cuentas de agarosa de proteína A, los adsorbentes de membrana de proteína A pueden acelerar significativamente el aislamiento a escala de laboratorio de anticuerpos y otras proteínas que expresan fragmentos de Fc. Los métodos apropiados de análisis y cuantificación pueden acelerar aún más el procesamiento de proteínas, lo que permite que el aislamiento / caracterización se complete en un día de trabajo, en lugar de más de 20 horas de trabajo.

Aislamiento Y Caracterización De Proteínas Quiméricas Humanas Que Expresan Fc Usando Adsorbentes De Membrana De Proteína A Y Un Flujo de Trabajo Optimizado

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

isolation-characterization-chimeric-human-fc-expressing-proteins

Research

JoVE Journal

Bioengineering

7.1K vistas.  Ohio University. En comparación con la cromatografía de afinidad tradicional utilizando columnas empaquetadas de cuentas de agarosa de proteína A, los adsorbentes de membrana de proteína A pueden acelerar significativamente el aislamiento a escala de laboratorio de anticuerpos y otras proteínas que expresan fragmentos de Fc. Los métodos apropiados de análisis y cuantificación pueden acelerar aún más el procesamiento de proteínas, lo que permite que el aislamiento / caracteriza...

Video: Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creación de dos dimensiones con dibujos sustratos para el confinamiento de proteínas y células

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Investigación

Bioingeniería

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

La detección y aislamiento de las células de melanoma circulantes mediante Flujometría fotoacústica

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrana-COLUMNA: una herramienta bioquímica para identificar las interacciones proteína-proteína de Proteínas de la Membrana<em&gt; En Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Síntesis de una proteína artificial mediada Intein hidrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Caracterización de Sistemas Complejos Uso del Diseño de Experimentos Enfoque: Expresión de Proteínas transitoria en tabaco como un estudio de caso

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Expresión, aislamiento y purificación de soluble e insoluble Biotinylated Proteínas del Tejido Nervioso para regeneración

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

La luminiscencia de energía de resonancia de transferencia para estudiar cambios conformacionales en proteínas de membrana expresado en células de mamífero

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Producción, Caracterización y posibles usos de un Humano esofágica mucosas Modelo ingeniería tisular 3D

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Caracterización biomecánica de los tejidos blandos humanos utilizando sangría y Ensayo de tracción

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabricación y caracterización de tejido óptico fantasmas que contiene macroestructura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...