Os autores desejam agradecer ao Sr. Luis F. Delgadillo (Departamento de Engenharia Química e Biomolecular da Universidade de Ohio), Ao Sr. Matthew H. Williams (Departamento de Engenharia Química e Biomolecular da Universidade de Ohio) e ao Dr. Filiberto Cedeno-Laurent (Brigham and Women's Hospital e Harvard Medical School) pela assistência técnica especializada. Os autores também desejam agradecer aos alunos do Winter 2011 CHE 404/BME 504 e Outono 2012 CHE 4830/BME 5830 (Departamento de Engenharia Química e Biomolecular e Programa de Engenharia Biomédica da Universidade de Ohio) por aconselhamento técnico e discussão. Este trabalho foi apoiado pela National Science Foundation (NSF) Major Research Instrumentation grant CBET-1039869 (MMB), NSF CBET-1106118 (MMB), Dermatology Foundation Research Grant A050422 (SRB), National Institutes of Health (NIH) NCI grant 1R15CA16 1830-01 (MMB), NIH Kirschstein-NRSA Pós-Doutorado F32CA144219-01A1 (SRB), NIH/NCI R01CA173610 (CJD) e NIH NCCAM grant R01AT004628 (CJD).

1. Verifique a afinidade pela proteína A
<ol><li>Verifique se a proteína de expressão de Fc (proteína de fusão recombinante ou anticorpo IgG, até então referido como "proteína") tem afinidade aceitável para a proteína A14-17 antes de usar a proteína A adsorber de membrana, e teste para a presença da proteína na solução de estoque ( porexemplo, cultura celular supernante clarificada por centrifugação a 1.000 x g por 5 min para pelotas células suspensas). Use citometria de fluxo, mancha ocidental, ELISA, etc. para detectar função proteica e/ou presença de Fc, com base na preferência do laboratório. Idealmente, quantifique a proteína na cultura celular supernascida (passo 6) para evitar exceder a capacidade de adsorber de membrana A. Proceda se a proteína for detectada.</li>
</ol>2. Prepare proteína Um Adsorber de Membrana e Supernatante da Cultura Celular
<ol><li>Siga as instruções do fabricante para preparar o adsorber de membrana. Nunca deixe o ar entrar no adsorber da membrana. Todas as soluções perfundidas através do adsorber de membrana devem estar em temperatura ambiente e pré-filtradas usando um filtro de 0,22 μm. Encha uma seringa de 10 ml com 0,22 μm filtrado de soro fisofado de Dulbecco (DPBS) ou tampão de opções e bolhas de ar de descarga. Perfuse DPBS para remover a solução de armazenamento e equilibrar o adsorber de membrana imediatamente antes do uso.</li>
	<li>Filtre a cultura celular sobrenatante imediatamente antes da perfusão através do adsorber de membrana, usando uma unidade de filtragem estéril de 0,22 μm acionada a vácuo.</li>
</ol>3. Carregue a proteína A Membrana Adsorber
<ol><li>Cultura celular aspirada sobrenatante em uma seringa Luer Lock (30 ml ou maior), e descarregar quaisquer bolhas de ar.</li>
	<li>Monte materiais e equipamentos como mostrado na Figura 1. Conecte a seringa à entrada do adsorber da membrana. Conecte tubos flexíveis à saída de adsorber de membrana. Se desejar, coloque um filtro de seringa de 0,22 μm entre a seringa e o adsorber de membrana. Use um frasco ou garrafa para pegar o fluxo (também conhecido como filtrado), a partir do adsorvo.</li>
	<li>Ajuste a bomba de seringa na taxa de fluxo volumoso desejada, mas não exceda a taxa de fluxo recomendada pelo fabricante (por exemplo, 10 ml/min). Perfunda a cultura celular supernacida através do adsorber de membrana, coletando fluxo através de um béquer ou outro recipiente. Recarregue a seringa com supernacantes necessários.</li>
	<li>Se desejar, teste o fluxo através da presença de proteínas usando citometria de fluxo, mancha ocidental, ELISA, etc. com base na preferência do laboratório. Normalmente é desnecessário reutilizar o fluxo.</li>
</ol>4. Proteína elute do Adsorber de Membrana
<ol><li>Lave o adsorvo de membrana com 10 ml DPBS para remover qualquer proteína não ligada.</li>
	<li>Proteína eluto do adsorber de membrana na taxa de fluxo desejada (por exemplo, 1 ml/min) usando 10-15 ml de tampão de elução(por exemplo, tampão de elução à base de amina (pH 2.8)). Capturar elunato em um tubo contendo tampão neutralizante (por exemplo, 1 M Tris (pH 9.4)) a 10% do volume de elução (1,0-1,5 ml).</li>
	<li>Alternativamente, a proteína eluto em um ml incrementa em tubos contendo 100 μl de tampão de neutralização, usando um volume total de 10-15 ml tampão de eluição. Use o método de caracterização preferido para testar cada fração para a presença de proteína.</li>
	<li>Regenerar o adsorber de membrana de acordo com as instruções do fabricante. Perfuse 10 ml de DPBS filtrado de 0,22 μm ou tampão de escolha, depois 10 ml de 0,22 μm filtrado 50 mM NaOH em 1 N NaCl, e finalmente 10 ml de DPBS filtrados de 0,22 μm. Encha o adsorber de membrana com 20% de etanol em DPBS para armazenamento a longo prazo a 4 ºC.</li>
</ol>5. Concentre e digele a proteína
<ol><li>Deposite todas as frações de elunato (ou frações de elução contendo proteínas conforme determinado na etapa 4.3) em uma unidade de filtro centrífugo de corte de peso molecular de 10 kDa. Siga as instruções do fabricante para centrifugação.</li>
	<li>Dique o retentato em uma pequena unidade de diálise de volume (corte de peso molecular de 10 kDa) contra o tampão de escolha, seguindo as instruções do fabricante. O buffer pode precisar ser adicionado ao material dialisado se a precipitação de proteína for uma preocupação. Se desejar, o material dialisado pode ser refrigerado até que a etapa 6 possa ser realizada, mas o armazenamento a longo prazo sem conservantes não é recomendado.</li>
</ol>6. Quantifique e caracterize o produto purificado em um procedimento de mancha ocidental acelerado
<ol><li>Resolva as normas de proteína purificada e Fc no gel de escolha pela SDS-PAGE, sob condições de redução ou não de18-20. Use um mini gel em vez de gel midi para minimizar o tempo de execução.<ol>
<li>Se ainda não for conhecido, determine a faixa de padrões de Fc sobre qual sinal de banda varia linearmente em relação à quantidade de carga(por exemplo, 0,1-1 mgs). Use o mesmo gel, condições de funcionamento, condições de transferência e reagentes que serão usados para quantificar e caracterizar proteínas purificadas.</li>
			<li>Carregue várias quantidades amostrais de proteína purificada, incluindo amostras diluídas com DPBS, para garantir que os sinais de banda de proteína purificada estejam dentro da faixa de sinal linear dos padrões de Fc na análise de imagens.</li>
		</ol>
</li>
	<li>Transfira proteínas do gel para uma membrana de flúor de polivinilidene (PVDF) em um sistema tampão descontínuo usando uma mancha semidry13, seguindo as instruções do fabricante da mancha. O tempo de transferência é tipicamente de 5-10 min. Se desejar, Coomassie manche o gel após a transferência para verificar a eficiência da transferência21.</li>
	<li>Realize a imunoblotação com anti-Fc ou anti-IgG conjugado para fosfattase alcalina (AP) ou peroxidase de rabanete de cavalo (HRP), com base na preferência do laboratório, usando um sistema de detecção de proteína assistida a vácuo. O tempo de imunoblotting é tipicamente inferior a 1 hora.</li>
	<li>Desenvolver imunoblot usando o substrato e o método de escolha (por exemplo, substrato AP e chemiluminescence aprimorada).</li>
	<li>Quantifique a proteína purificada por análise de imagem. Realize uma regressão linear nos sinais de banda dos padrões fc. Se R2>0,90, use a equação para a linha para calcular a quantidade de proteína de seu sinal de banda(s). Conta para diluição amostral, se necessário. Uma vez que a quantificação da proteína é realizada com base em Fc, ajuste o valor para diferenças de peso molecular entre a proteína e Fc. Se R2<0,90, repita o passo 6 em sua totalidade.</li>
	<li>Validar a proteína usando ensaios funcionais ou métodos para detectar Fc via citometria de fluxo, mancha ocidental, ELISA, etc. com base na preferência do laboratório.</li>
	<li>Diluir a proteína à concentração desejada e/ou adicionar quaisquer conservantes ou estabilizadores desejados à solução de proteína purificada antes de aliquotar para armazenamento a longo prazo, congelado ou não.</li>
</ol>

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Não há nada para revelar.

O protocolo aqui descrito foi desenvolvido para isolar rapidamente e caracterizar uma proteína de fusão quimrica expressando Fc humano (Gal-1hFc), sem comprometer significativamente a recuperação do produto ou o custo inflacionário. Os principais componentes do fluxo de trabalho simplificado são um adsorber de membrana A para isolamento, e transferência semidária e imunoblotação assistida a vácuo para caracterização por manchas ocidentais.
A dramática diminuição do tempo de pr...

Isolamento de anticorpos e proteínas recombinantes expressando imunoglobulina G (IgG) Fragmentos de fc de supernantes da cultura celular e soluções celulares diluídas podem ser realizados usando cromatografia de afinidade da proteína A. Em laboratórios básicos de ciência e engenharia e indústria, colunas repletas de proteína porosa A-coated agarose, vidro ou contas poliméricas são mais comumente usadas para cromatografia de afinidade, apesar dos altos custos financeiros e dos longos tempos de processamento1-3. É bem apreciado que a cromatografia de afinidade representa o maior gargalo de gastos e processamento em ambientes laboratoriais e industriais2,4. Como resultado, inúmeras melhorias foram desenvolvidas para diminuir o custo e o tempo de processamento sem sacrificar a recuperação geral do produto do trem de processo1,2,5-7. Particularmente promissor para o isolamento de anticorpos e outras proteínas que expressam Fc é a cromatografia de afinidade usando uma proteína A de adsorber de membrana2,5,7-10. Considerando que a captura de Fc na cromatografia da coluna é limitada à difusão (ou seja, a proteína que expressa fc deve difundir dentro e através de poros internos para atingir a maioria da proteína A) com queda de alta pressão através da coluna, o transporte de massa em adsorbers de membrana é impulsionado por convecção a granel, resultando em evitar limitações de difusão8,9,11,12. Além disso, a queda de pressão no adsorber da membrana é baixa, permitindo assim taxas de fluxo de perfusão mais rápidas em comparação com as colunas embaladas por contas8,9,11,12. Assim, para o isolamento em escala laboratorial, prevê-se que a cromatografia da membrana reduza o tempo de isolamento em várias horas e aumente a captura do produto em comparação com a cromatografia da coluna. Além disso, foram propostos modelos matemáticos para adsorção de IgG em adsorbers de membrana8,9,11,12, permitindo assim que os usuários finais prevejam o desempenho em laboratório.
Outro gargalo nos laboratórios básicos de ciência e engenharia é a caracterização da proteína de expressão de Fc. No entanto, a seleção de métodos apropriados para completar ensaios de caracterização, tais manchas ocidentais, pode reduzir muito o tempo gasto em testes de produtos. Por exemplo, a transferência semidária em um sistema tampão descontínuo pode realizar a transferência eletroforética de proteínas de um gel SDS-PAGE para uma membrana de difluoredina polivinina (PVDF) em questão de minutos, em vez de 1-2 horas de transferência de tanque (molhado) em um sistema tampão contínuo13.
Um protocolo rápido, barato e eficaz para isolar e caracterizar uma proteína de fusão quimrica expressando um fragmento fc de IgG humano é descrito aqui. A maioria dos equipamentos está prontamente disponível em laboratórios de ciência biomédica básica padrão, biologia, química e (bio)engenharia química, e todos os reagentes estão disponíveis comercialmente. Embora os resultados representativos tenham sido gerados a partir do isolamento e caracterização de uma proteína de fusão quimrica murina galectin-1/human Fc (Gal-1hFc), o protocolo simplificado pode ser aplicado ao isolamento de outras moléculas que possuem um fragmento de Fc, como anticorpos, fragmentos de Fc ou outras proteínas de fusão fc. Nossas melhorias diminuíram drasticamente o tempo de processamento (apenas 4 horas, mas mais tipicamente ~9 horas, em comparação com mais de 20 horas de trabalho) ao mesmo tempo em que alcançaram recuperação de produtos semelhantes ou melhoradas em comparação com os métodos padrão.

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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>
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			<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>
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			<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

A escala laboratorial para a purificação em escala industrial de biomoléculas de supernacantes de cultura celular e soluções celulares límedas pode ser realizada usando cromatografia de afinidade. Enquanto a cromatografia de afinidade usando proteína porosa As contas de agarose embaladas em colunas é sem dúvida o método mais comum de isolamento em escala laboratorial de anticorpos e proteínas recombinantes expressando fragmentos de Fc de IgG, pode ser um processo demorado e caro. As restrições de tempo e financeiros são especialmente assustadoras em pequenos laboratórios básicos de ciência que devem recuperar centenas de microgramas para quantidades miligramas de proteína de soluções diluídas, mas não têm acesso a sistemas de entrega de líquidos de alta pressão e/ou pessoal com experiência em bioseparações. Além disso, a quantificação e caracterização do produto também podem alongar excessivamente o tempo de processamento ao longo de vários dias úteis e inflar despesas (consumíveis, salários, etc.). Portanto, um protocolo rápido, barato, mas eficaz é necessário para o isolamento em escala laboratorial e caracterização de anticorpos e outras proteínas que possuam um fragmento de Fc. Para isso, elaboramos um protocolo que pode ser preenchido por equipe técnica de experiência limitada em menos de 9 horas (aproximadamente um dia útil) e tão rapidamente quanto 4 horas, em oposição aos métodos tradicionais que exigem mais de 20 horas de trabalho. A maioria dos equipamentos necessários está prontamente disponível em laboratórios de ciência biomédica padrão, bioquímica e (bio)engenharia química, e todos os reagentes estão disponíveis comercialmente. Para demonstrar este protocolo, são apresentados resultados representativos nos quais a galepina quimrica murina-1 fundida ao DFc humano (Gal-1hFc) da cultura celular foi isolada usando um adsorber de membrana A da proteína A. Gal-1hFc purificado foi quantificado por meio de um procedimento acelerado de análise de manchas ocidentais e caracterizado por citometria de fluxo. O fluxo de trabalho simplificado pode ser modificado para outras proteínas que expressam Fc, como anticorpos, e/ou alteradas para incorporar métodos alternativos de quantificação e caracterização.

O protocolo simplificado para isolamento e caracterização de proteínas que expressam Fc é rotineiramente usado para processar galectina-1 murina quimrica fundida ao Fc humano (Gal-1hFc) de supernantes de cultura celular diluída. O fluxograma na Figura 2 ilustra o fluxo de trabalho e o tempo para cada passo no protocolo. Para um lote típico de 300 ml de supernascimento, o tempo total de processamento é de aproximadamente 9 horas quando o teste opcional de citometria de fluxo de frações de eluçã...

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

Em comparação com a cromatografia de afinidade tradicional usando colunas repletas de proteínas Agarose, os adsorbers de membrana A da proteína A podem acelerar significativamente o isolamento em escala laboratorial de anticorpos e outras proteínas que expressam fragmentos de Fc. Os métodos adequados de análise e quantificação podem acelerar ainda mais o processamento de proteínas, permitindo que o isolamento/caracterização seja concluído em um dia útil, em vez de mais de 20 horas de trabalho.

Isolamento e caracterização de proteínas quimricas que expressam fc humanos usando adsorbers de membrana de proteína e um fluxo de trabalho simplificado

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 visualizações.  Ohio University. Em comparação com a cromatografia de afinidade tradicional usando colunas repletas de proteínas Agarose, os adsorbers de membrana A da proteína A podem acelerar significativamente o isolamento em escala laboratorial de anticorpos e outras proteínas que expressam fragmentos de Fc. Os métodos adequados de análise e quantificação podem acelerar ainda mais o processamento de proteínas, permitindo que o isolamento/caracterização seja concluído em um dia útil, em...

Vídeo: 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.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

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

Bioengenharia

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.

Detecção e isolamento de células de melanoma circulantes usando Fluxometria 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-COLUNA: Uma Ferramenta bioquímicos na identificação de interações proteína-proteína de Proteínas de Membrana<em&gt; In 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.

Síntese de uma proteína artificial intein mediada 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.

Caracterização de Sistemas Complexos Usando o Projeto de Experimentos Abordagem: Transient Protein Expression em tabaco como um Estudo 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.

Expressão, isolamento e purificação do solúveis e insolúveis biotinilados Proteínas de Tecido Nervoso Regeneração

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.

Luminescência Ressonância transferência de energia para estudar mudanças conformacionais na membrana proteínas expressas em células de mamíferos

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.

Produção, Caracterização e usos potenciais de uma mucosa esofágica Modelo 3D Human Tissue-engenharia

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.

Caracterização biomecânica de tecidos moles Humanos Usando recuo e testes de tração

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.

Fabricação e caracterização de espectros ópticos de tecido contendo Macrostructure

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