Este trabalho foi financiado pelos Institutos Nacionais de Saúde (DP1CA174426 para Carlos F. Barbas) e da Universidade ShanghaiTech, Shanghai, China (a JL). Gráficos moleculares foram gerados utilizando pymol.

1. Clonagem <ol><li> Obter de dois dedos domínios ZIF substituído por alanina que foram sub-clonados no sistema de expressão vector pET-28 e estão disponíveis mediante pedido (PET-2F-ZiF). 34 </li><li> EmGFP amplificar por PCR a partir do plasmídeo pBAD-esmeralda com os iniciadores 5 &#39;Xmal-EmGFP (3-ATGGTGAGCAAGGGCGAGGAGCTGTTCAC 5&#39;-GGAAATTG CCCGGG&#39;; sítio Xmal em negrito) e 3 &#39;Saci-EmGFP (5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3&#39; site eu Sac em negrito);. <ol><li> Use 5 ng de ADN molde, 10 ul de tampão de polimerase 10x, 1 Unidades (U) de Taq DNA polimerase, 0,2 mM de cada dNTP e 0,2 pM de cada iniciador numa solução de 100 uL com o restante volume constituídos de água destilada / desionizada . Use as condições dos ciclos: 95 ° C durante 5 min; 30 ciclos de 95 ° C durante 30 seg, 55 ° C durante 30 seg e 72 ° C durante 1 min; 72 ° C durante 10 min. Purifica-se o produto de PCR por gel extractie em determinar a concentração de ADN utilizando um espectrofotómetro de medição Abs 260 x 50 ug / ml. </li></ol></li><li> Digest pET-2F-ZiF e a inserção que codifica EmGFP com as enzimas de restrição Xmal e Sac I em tampão recomendado durante 3 horas a 37 ° C utilizando 10 U de enzima por 1 ug de ADN. Visualizar o ADN por electroforese em gel de agarose, utilizando um corante fluorescente intercalante, tal como brometo de etídio. </li><li> Purifica-se o ADN digerido por gel extraction kit e determinar a concentração de DNA por um espectrofotómetro Abs medindo 260 x 50 ug / ml. </li><li> Ligadura o ADN que codifica EmGFP purificado em 50-100 ng de pET-2F-ZiF usando 1 U de T4 DNA ligase durante pelo menos 1 h à TA. Para melhores resultados, realizar a reacção de ligação utilizando uma razão de 6: molar de inserção-para-um vector. </li><li> Descongelar 50 ul de quaisquer células XL-1 azul de E. coli quimicamente competentes de Escherichia em gelo e misturar suavemente com 10-20 ng de ligado pET-2F-ZiF-EmGFP. </li><li> Manter em gelo durante 30 min. O choque térmico a mistura a 42 ° C durante 90 seg e recuperar as células em 2 ml de caldo super óptima com repressão catabólica (SOC) durante 1 hora a 37 ° C com agitação. </li><li> Espalhe 100 ul de cultura de recuperação em um caldo lisogenia (LB) placa de ágar com 50 ug / ml de canamicina e incubar O / N a 37 ° C. </li><li> No dia seguinte, inocula 6 ml de Super Broth (SB) ou cultura LB contendo 50 ug / ml de canamicina com uma colónia da placa de agar LB e a cultura O / N a 37 ° C. Nota: Colony PCR utilizando os iniciadores 5 &#39;e Xmal-EmGFP 3&#39; Saci-EmGFP poderia ser usado para identificar os clones positivos antes de miniprep. </li><li> Purificar pET-2F-ZiF-EmGFP por miniprep e confirmar plasmídeo por sequenciação de ADN utilizando o promotor de T7 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;). </li></ol> 2. Expressão e purificação <ol><li> Descongelar 50 ul de quimicamente competentes BL21 de E. coli células no gelo e misture delicadamente com 100 ng do PET-2F-ZiF-EmGFP plasmid. Transformação como descrito nos passos 1,7-1,8. </li><li> No dia seguinte, inocular 10 ml de meio LB contendo 50 ug / ml de canamicina com uma única colónia e crescer O / N a 37 ° C com agitação. </li><li> No dia seguinte, dilui-se os 10 ml da cultura D / N para 1 L de meio LB suplementado com 50 ug / ml de canamicina, 0,2% de glucose e 100 mM de ZnCl2. Crescer a cultura a 37 ° C com agitação até uma densidade óptica a 600 nm (OD 600) de 0,8 e induzir a expressão da proteína com 2 mM de isopropil-β-D-1-tiogalactopiranósido (IPTG). Após 6 horas de crescimento a 37 ° C, as células da colheita por centrifugação a 5000 xg durante 10 min a 4 ° C. Nota: As condições de indução são muito variáveis ​​e dependem da estabilidade das proteínas a ser expressa. Monitorar a OD a 600 a cada 30 min até uma DO600 de 0,8 seja alcançado. </li><li> Ressuspender o sedimento celular em 5 ml de tampão de lise (50 mM Tris-HCl, 500 mM de NaCl, 100 mM de ZnCl2, 1 mM ditiotreitol (DTT), 1 mM de MgCl 2, 1 mM de fluoreto de fenilmetilsulfonilo (PMSF) e 10 mM de imidazol, pH 8,0). Lisar as células no gelo por ultra-sons com a seguinte configuração: 50% de potência, 4 min de tempo de processo com 5 sec on / off intervalos de 10 segundos. Evitar o sobreaquecimento da solução. </li><li> Centrifuga-se o lisado celular a 25000 xg durante 30 min a 4 ° C e transferir o sobrenadante para um tubo de recolha de fresco. Para melhores resultados, executar todos os passos seguintes a 4 ° C. </li><li> Executar o sobrenadante através de uma coluna pré-embalada com 1 ml de suspensão equilibrada de Ni-NTA. Lava-se a resina com 20 ml de tampão de lavagem (50 mM Tris-HCl, 500 mM de NaCl, 100 fiM de ZnCl2, 1 mM de DTT, 1 mM de MgCl 2 e 30 mM de imidazol, pH 8,0). </li><li> Elui-se a proteína com 5 ml de tampão de eluição (50 mM Tris-HCl, 500 mM de NaCl, 100 fiM de ZnCl2, 1 mM de DTT, 1 mM de MgCl2, e 300 mM de imidazole, pH 8,0). </li><li> Tampão de trocar a proteína eluída com tampão de armazenamento (50 mMTris-HCl, 500 mM de NaCl, 100 mM de ZnCl2, 1 mM de DTT, 1 mM de MgCl2 e glicerol a 10%, pH 8,0) e concentrar a proteína de pelo menos 40 jiM utilizando um concentrador de rotação por centrifugação de acordo com as instruções do fabricante. </li><li> Determinar a concentração de proteína pelo ensaio de Bradford ou BCA. Misturar 2 ul de proteínas purificadas com 2 ul 2 × SDS-PAGE de corante de carga, ferver a 95 ° C durante 10 min e em seguida resolver a 4% -20% de Tris-Glicina SDS-PAGE para avaliar a pureza de proteína (Figura 2). </li></ol> 3. Proteína de armazenamento <ol><li> Alíquota da proteína concentrada, congelamento de flash em nitrogênio líquido e armazenar a -80 ° C. Para as proteínas de fusão EmGFP, cobrir o tubo com folha de alumínio para proteger a proteína da luz. </li><li> Evite congelamento e descongelamento da solução de proteína repetido. Nota: ZiF proteínas fundidas são estáveis ​​sob estas condições durante pelo menos 1 mês. Ou armazenamento inadequado mês&gt; 4 pode levara precipitação de proteínas ou de fotobranqueamento EmGFP. </li></ol> Transdução 4. Protein <ol><li> Manter as células HeLa em meio de Eagle modificado por Dulbecco (DMEM) contendo 10% (v / v) de soro fetal bovino (FBS) e 1% de antibiótico-antimicótico a 37 ° C em uma atmosfera totalmente humidi ficada com 5% de CO 2. Nota: As células passadas mais de 30 vezes não são recomendados para a transdução de proteína. </li><li> Pré-revestimento de uma placa de 24 poços com 500 ul de 50 ug / ml de poli-lisina para 30 a 60 min a 25 ° C. Semear as células num prato de 24 cavidades a uma densidade de 2 x 10 5 células por poço. Em 24 horas após a sementeira, ou uma vez que as células são entre 80% -90% confluentes, retirar o suporte de cada poço e lava-se com 500 ul de DMEM isento de soro pré-aquecido (SFM). </li><li> Para cada poço, adicione 250 mL de SFM contendo 2 uM de proteína ZiF-EmGFP e 100 uM de ZnCl2. Incubar as células a 37 ° C durante 1 h. Nota: domínios ZIF entrar nas células primarily através de macropinocitose, 34, o qual é um mecanismo dependente de energia. Portanto, as células devem ser incubados a 37 ° C durante internalização proteína eficiente. </li><li> Remover meios a partir de células e lava-se três vezes com 500 ul de cálcio e sem magnésio salina tamponada com fosfato de Dulbecco (DPBS) suplementado com 0,5 mg / ml de heparina. Nota: A heparina é necessário remover a proteína ligada à superfície que pode complicar análises a jusante. </li><li> (Opcional) tratamentos repetidos até três vezes para maior entrega. </li><li> Isolar células tratadas imediatamente depois de heparina de lavagem por digestão com 0,05% de tripsina-EDTA a 37 ° C durante 2 min. Re-suspender as células isoladas em 0,5 ml de DPBS suplementadas com 1% de FBS. </li><li> Medir a intensidade de fluorescência de cada amostra por 35 citometria de fluxo utilizando o isotiocianato de fluoresceína (FITC) canal (Figura 3). Ajuste a dispersão para a frente (FSC) e dispersão lateral (SSC) para colocar a população deinteresse na escala, assegurando que as populações com propriedades diferentes são resolvidos um do outro. </li><li> Colete 10.000 eventos ao vivo para cada amostra e analisar os dados utilizando citometria de fluxo de análise de dados software. 36 Normalize fluorescência de células HeLa tratadas com ZiF-EmGFP a essas células tratadas apenas com SFM. </li></ol>

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Aqui, um protocolo passo-a-passo para a entrega de proteína utilizando dedo de zinco (ZiF) domínios de células-permeável é apresentado. O domínio ZiF não reduz a atividade de carga enzimática fundido 34; permite a produção e purificação de proteínas em rendimentos quase idênticos aos observados com a proteína não modificada; e pode transportar proteínas e enzimas para uma ampla gama de tipos de células, com as eficiências que excedem os sistemas tradicionais de penetração de células de p...

Altamente estratégias de fornecimento de proteína eficientes e versáteis são críticos para muitos pesquisa básica e aplicações terapêuticas. A entrega directa das proteínas purificadas em células representa um dos métodos mais seguros e mais fáceis para alcançar este objectivo. 1,2 Ao contrário das estratégias que dependem de expressão de genes de ácidos nucleicos, 3-5 entrega proteína não apresenta nenhum risco de mutagénese de inserção, é independente do transcrição / máquinas e tradução celular permite um efeito imediato. No entanto, a falta de métodos simples e generalizáveis ​​para dotar a atividade de penetração celular para proteínas confunde rotineiramente sua entrada direta nas células. Os métodos actuais para facilitar a entrega intracelular de proteínas são baseadas no uso de ocorrência natural ou 6-8 concebidos de penetração de células, péptidos 9-12 domínios de transdução de sobrealimentação, 13,14 nanopartículas 15, 16 e lipossomas partículas semelhantes a vírus 17,18 </sup&gt; e materiais de microesferas poliméricas. 19 Infelizmente, muitas dessas abordagens são dificultados pelas taxas de captação celular baixos, 20,21 pobre estabilidade, 22 inadvertida tipo de célula de especificidade, 23 propriedades de escape baixo endossomais 24 e toxicidade. 25 Além disso, muitos proteína tecnologias de transdução de reduzir a bioactividade das proteínas entregues 14. O nosso laboratório demonstrou anteriormente que zinc-finger nuclease proteínas (ZFN) - endonucleases de restrição quimérico que consiste de uma Cys His 2 2 zinc-finger programável proteína de ligação a ADN e domínio de clivagem da endonuclease de restrição FokI 26-28 - são inerentemente célula.dia permeável. 29 Esta actividade de penetração celular surpreendente mostrou ser uma propriedade intrínseca do domínio dedo-de-zinco personalizados, com uma plataforma de ligação de ADN que surgiu como uma ferramenta poderosa para o genoma segmentado enengenha-, 30-32 e ser considerada como o resultado da constelação de seis resíduos carregados positivamente na superfície da proteína. De facto, várias proteínas de ligação de ADN, incluindo c-Jun e N-DEK foram mostrados para possuir uma capacidade inata para atravessar as membranas celulares. 33 Mais recentemente, no nosso laboratório expandido estes resultados e demonstraram que a actividade da célula de penetração de zinco finger (ZIF) da domínios poderia ser aproveitado para a entrega de proteínas intracelulares. Fusão genética de ambos domínios ZIF de um ou dois dedos para carga proteína específica levou a absorção eficiências que superaram muitos sistemas de entrega de peptídeos convencionais de penetração celular. 34 Mais notavelmente, entrega ZiF mediada não comprometeu a atividade de carga enzimática fundido e facilitado altos níveis de entrega cytosolic. Colectivamente, estes resultados demonstram o potencial do domínio ZiF para facilitar a entrega eficiente e fácil de proteínas, e potencialmente mais diversos tipos de macromoléculas, em células. Aqui, um protocolo detalhado passo-a-passo sobre como implementar a tecnologia ZiF para entrega de proteína em células de mamíferos é apresentada. Nós previamente construído um conjunto de um, dois, três, quatro, cinco e seis domínios dedo-Zif que não possuem a capacidade de se ligar ao ADN, devido a substituição de cada um dos resíduos de ligação ao ADN de hélice α, mas são capazes de fornecer proteínas em células 34 (Figura 1). A produção e a transdução de esmeralda GFP (EmGFP) em células HeLa usando um domínio ZiF de dois dedos é descrito. Este protocolo é extensível para quase qualquer proteína solúvel capaz de expressão em Escherichia coli e quase qualquer tipo de células de mamíferos. Os resultados esperados são fornecidos e estratégias para maximizar o desempenho do sistema também são discutidos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Dois dedos de proteínas de fusão ZiF-EmGFP pode ser expresso em E. coli com&gt; 95% de homogeneidade e rendimentos elevados (&gt; 25 mg / ml) (Figura 2). Em geral, de um e de dois dedos proteínas de fusão ZiF pode ser produzido em quantidades quase idênticas às de proteína não modificada tipo selvagem. No entanto, em alguns contextos, as proteínas de fusão ZiF cinco e seis dedos não são capazes de ser produzidos em rendimentos elevados o suficiente para aplicações a jusante. <...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Domínios dedo-de-zinco são intrinsecamente célula-permeável e capaz de mediar a entrega de proteína em uma ampla gama de tipos de células de mamíferos. Aqui, um protocolo detalhado passo-a-passo para a implementação da tecnologia de dedo de zinco para a entrega intracelular de proteínas é apresentada.

Entrega Protein direto para células de mamíferos utilizando células-permeável Cys<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt; Domínios zinc-finger

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Research

JoVE Journal

Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

9.8K visualizações.  The Scripps Research Institute.  Domínios dedo-de-zinco são intrinsecamente célula-permeável e capaz de mediar a entrega de proteína em uma ampla gama de tipos de células de mamíferos. Aqui, um protocolo detalhado passo-a-passo para a implementação da tecnologia de dedo de zinco para a entrega intracelular de proteínas é apresentada. 

Vídeo: Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Longo prazo células de mamíferos utilizando imagens do Largo-Campo Microscopia

Biologia

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing e Subcultura células de mamíferos

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Engenharia de Proteínas Cell-permeáveis

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery Fase Líquida

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

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genoma Edição com CompoZr personalizados Nucleases dedo de zinco (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Screening siRNA identificar Ubiquitin e ubiquitina-como reguladores do sistema de caminhos biológicos em células de mamíferos cultivadas

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Identificação de parceiros de interação de proteínas em células de mamíferos utilizando SILAC-imunoprecipitação quantitativos Proteômica

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

Zinc-finger nucleases Gene maior focalização em células estaminais embrionárias humanas

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Exame de proteínas ligadas ao DNA em células de mamíferos Nascente usar a técnica de-BrdU-chip-Slot Ocidental

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...