Os autores gostariam de agradecer a Universidade de Akron para o financiamento que apoiaram este trabalho.

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Engenharia de proteínas recombinantes é uma técnica muito poderosa que se estende por muitas disciplinas. É rentável, sintonizável e um procedimento relativamente simples, permitindo a produção de altos rendimentos de proteínas personalizados. É importante notar que a concepção e expressão de proteínas-alvo nem sempre é directa. Expressão basal e estabilidade da proteína recombinante depender de escolhas específicas de vetoriais, E. estirpes de E. coli, células de adições marca pep...

Proteínas de cobrir uma vasta gama de biomoléculas que são responsáveis ​​por muitas funções biológicas, levando à formação de tecido adequado e organização. Estas moléculas de milhares de iniciar as vias de sinalização que controlam a regulação positiva e / ou sub-regulação de genes e outras proteínas, mantendo o equilíbrio dentro do corpo humano. O rompimento de uma única proteína afeta toda esta teia de sinais, que podem levar ao aparecimento de distúrbios ou doenças devastadoras. Engenharia proteínas individuais no laboratório oferece uma solução para combater estes efeitos adversos e oferece uma alternativa aos medicamentos de pequenas moléculas. Em 1977, um gene que codifica a sequência de aminoácidos de somatostatina 14 foi um dos primeiros polipeptídeos engenharia criadas utilizando E. coli 1. Logo depois, em 1979, a insulina foi clonado no plasmídeo pBR322, transformada, expressa, purificada e 2. Desde então, as proteínas recombinantes têm expandido sua influência em vários campos da research como biomateriais, a entrega da droga, engenharia de tecidos, biofármacos, agricultura, enzimas industriais, biocombustíveis, etc (para revisões ver referências 3-8). Isto é principalmente devido à versatilidade que a técnica oferece através da adição de porções de aplicação de produtos químicos específicos ou sequências de proteínas para fins incluindo, mas não limitado a, a identificação das proteínas alvo, de estabilização e de purificação. Através da tecnologia de DNA recombinante, as proteínas recombinantes podem ser expressas em uma variedade de sistemas de hospedeiros eucariotas e procariotas, incluindo mamíferos, plantas, insectos, leveduras, fungos e bactérias. Cada host oferece diferentes vantagens e, normalmente, o melhor sistema é determinado com base na função da proteína, rendimento, estabilidade, custo total, e escalabilidade. Células de bactérias, muitas vezes não têm os mecanismos de modificação pós-traducionais que os anfitriões fornecem eucarióticas (ou seja, glicosilação, dissulfeto de transição, e<html> tc.) 5. Como resultado, os sistemas de mamífero e de insecto geralmente resultam em melhor compatibilidade e de expressão de proteínas eucarióticas, no entanto estes hospedeiros são geralmente mais caros e 9 demorado. Portanto, E. coli é o hospedeiro preferido para o nosso sistema de expressão, porque as células se expandem rapidamente em condições de crescimento de baixo custo e os mecanismos de expressão genética são bem compreendidos 5,9. Além disso, este sistema é de fácil aumento de escala para fins de produção e resulta em proteínas funcionais, apesar da ausência de modificações pós-translacionais 10. A E. coli K12 tensão é escolhida neste protocolo para a clonagem, porque esta estirpe oferece excelentes rendimentos plasmídeos baseados em alta eficiência de transformação. Além disso, uma E. coli estirpe BL21 utilizado para a expressão porque esta estirpe hospedeira contem o gene da RNA polimerase de T7, que proporciona a expressão da proteína e da estabilidade controlada 11. tenda &quot;&gt; Após a seleção anfitriã, mais cuidado deve ser tomado na escolha do vetor de expressão ideal para facilitar selecionados e controlados expressão da proteína. Sintetizando proteínas recombinantes começa com uma sequência de DNA alvo que é clonado sob a direção de transcrição bacteriófago T7 e sinais de tradução e expressão é induzida em células hospedeiras contendo cópias cromossómicas do gene da T7 ARN polimerase 12. Estes vectores, derivados de pBR322 plasmídeo vector (para uma revisão ver referência 13), é rigorosamente controlada pelo promotor T7 inicialmente desenvolvido por Studier e colegas 14 e fornecer adicional controlo por meio da inclusão do operador lac e o repressor lac (LAC1) 15,16. Para a engenharia de proteína recombinante, este sistema de expressão oferece a possibilidade de adaptar a uma sequência específica de aminoácidos de uma proteína desejada através da inserção de sequências de DNA alvo diferentes ou para criar proteínas de fusão composta de domínio combinados de proteínas individuais. Além disso, algumas séries de vetores incluem modificações tag peptídeo a ser colocados no N ou C terminal. Para os nossos propósitos de desenho, uma histidina (His) tag foi adicionado à sequência de ADN alvo para a purificação e uma sequência de 15 aminoácidos biotinylatable foi incluído para biotinilação 17,18. Neste protocolo de um plasmídeo contendo um gene de resistência à ampicilina, foi escolhido para realizar os nossos sequências de proteínas de fusão biotinylatable. A expressão é controlada por este vector, através do promotor lac de T7 e é facilmente induzida com isopropil β-D-1-tiogalactopiranosido (IPTG). Expressões de ensaio (culturas em pequena escala) são usados ​​para determinar a presença e a solubilidade da proteína alvo, que pode ser expresso numa forma solúvel ou insolúvel para formular os procedimentos de purificação. Uma proteína solúvel expressa dentro da célula bacteriana será submetido a dobragem espontânea para manter a sua estrutura nativa 19. Tipicamente, o nativoestrutura é termodinamicamente favorável. Em muitos casos, a actividade metabólica do hospedeiro não é favorável para a proteína alvo, colocando pressão no sistema que leva à produção de proteína insolúvel e a formação de corpos de inclusão na forma de agregados de proteínas insolúveis. Assim, a proteína alvo desnatura, tornando-os biologicamente inactivos, geralmente 20. Ambas as expressões de ensaio são incrementados, e os procedimentos de isolamento é determinada pela solubilidade da proteína alvo. Um adicional de renaturação ou redobramento etapa é necessária para as proteínas insolúveis. As proteínas recombinantes resultantes podem ser ainda purificado usando cromatografia de exclusão de tamanho. No casa de produção de proteína recombinante oferece vantagens de custo sobre os produtos comerciais desde miligramas de proteína alvo pode ser isolado por litro de cultura principal. A maior parte do equipamento necessário está disponível num laboratório biológico ou químico típico. Engenharia de proteínas permite a criaçãode proteínas de fusão de costume com funcionalidades adicionais que nem sempre estão disponíveis comercialmente. Figura 1 mostra os principais procedimentos envolvidos na engenharia de proteínas recombinantes. Com este sistema de expressão que criamos muitas proteínas biotinylatable, como o interferon-gama, fator de crescimento derivado das plaquetas e proteína morfogenética óssea-21-23, mas vamos nos concentrar em duas proteínas que nós projetamos para orientação do axônio, NGF (29 kDa ) e SEMA3A (91 kDa) 10 (para revisão ver referência 24). A biotinilação é uma técnica comum para a identificação, a imobilização e isolamento de proteínas marcadas utilizando a interacção biotina-estreptavidina bem conhecido 25-27. Sondas biofísicas 28,29, biossensores 30, e quantum dots 31 são alguns exemplos de sistemas que utilizam a alta afinidade de conjugação biotina-estreptavidina com um K d da ordem de 10 -15 M 27. A E. coli bioligase de estanho, BirA, auxilia na ligação covalente de biotina para a cadeia lateral de lisina encontrada no interior da sequência de biotina marcado 18,32. Tethering biotina aos materiais e biomoléculas produziu entrega sustentada de fatores de crescimento para a célula para múltiplas aplicações em engenharia de tecidos 21,33-35. Portanto, engenharia estas proteínas biotinylatable design personalizado é uma ferramenta poderosa que pode transcender a múltiplos interesses de pesquisa.

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">1,4-dithio--DL-threitol, DTT, 99.5%</td>
			<td style="width:191px;">Chem-Impex International</td>
			<td style="width:121px;">127</td>
			<td style="width:239px;">100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">2-Hydroxyethylmercaptan &#946;-Mercaptoethanol</td>
			<td>Chem-Impex International</td>
			<td>642</td>
			<td>250 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid, glacial</td>
			<td>EMD</td>
			<td>AX0073-9</td>
			<td>2.5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Bioshop</td>
			<td>AGR001.500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin sodium salt&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9518</td>
			<td>25 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antifoam 204</td>
			<td>Sigma-Aldrich</td>
			<td>A6426</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barstar-NGF pET-21a(+)</td>
			<td>GenScript USA Inc.</td>
			<td></td>
			<td>4 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BL21(DE3) Competent Cells</td>
			<td>Novagen</td>
			<td>69450</td>
			<td>1 ml; Expression Host</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bradford reagent</td>
			<td>Sigma-Aldrich</td>
			<td>B6916</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BugBuster</td>
			<td>Novagen</td>
			<td>70922-3</td>
			<td>100 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel filtration standard</td>
			<td>Bio-Rad</td>
			<td>151-1901</td>
			<td>6 vials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Bioshop</td>
			<td>GLY001.1</td>
			<td>1 L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">Guanidine hydrodioride amioformamidine hydrochloride</td>
			<td>Chem-Impex International</td>
			<td>152</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">His-Pur Ni-NTA Resin</td>
			<td>Thermo Scientific</td>
			<td>88222</td>
			<td>100 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>EMD&#160;</td>
			<td>HX0603-3</td>
			<td>2.5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole&#160;</td>
			<td>Chem-Impex International</td>
			<td>418</td>
			<td>250 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>Chem-Impex International</td>
			<td>194</td>
			<td>100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laemmli sample buffer</td>
			<td>Bio-Rad</td>
			<td>161-0737</td>
			<td>30 ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">Lauryl sulfate sodium salt, Sodium dodecyl surface</td>
			<td>Chem-Impex International</td>
			<td>270</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LB Broth&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NovaBlue Competent Cells</td>
			<td>Novagen</td>
			<td>69825</td>
			<td>1 ml; Cloning Host</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td>10 pack</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Chem-Impex International</td>
			<td>01247</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sema3A-pET-21a(+)</td>
			<td>GenScript USA Inc.</td>
			<td></td>
			<td>4 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td>1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886-1KG</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate diabasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136-500G</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5011</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Terrific Broth</td>
			<td>Bioshop</td>
			<td>TER409.5</td>
			<td>5 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetracycline hydrochloride</td>
			<td>Chem-Impex International</td>
			<td>667</td>
			<td>25 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris/Glycine/SDS Buffer, 10X</td>
			<td>Bio-Rad</td>
			<td>1610732</td>
			<td>1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone, pancreatic</td>
			<td>EMD</td>
			<td>1.07213.1000</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast extract, granulated</td>
			<td>EMD</td>
			<td>1.03753.0500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:299px;">Name of Kits/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:121px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">&#160;&#196;KTApurifier10</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">28-4062-64&#160;</td>
			<td>Includes kits and accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Benchtop Orbital Shaker</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:121px;">SHKE4000</td>
			<td>MAXQ 4000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">BirA500</td>
			<td style="width:191px;">Avidity</td>
			<td style="width:121px;">BirA500</td>
			<td style="width:239px;">Enzyme comes with reaction buffers and biotin solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dialysis Casette</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:121px;">66380</td>
			<td>Slide-A-Lyzer (Extra Strength)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dialysis Tubing</td>
			<td style="width:191px;">Spectrum Laboratories&#160;</td>
			<td style="width:121px;">132127, 132129</td>
			<td style="width:239px;">MWCO: 25,000 and 50,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Flow Diversion Valve FV-923</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">11-0011-70</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FluoReporter Biotin Quantification Assay Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:121px;">1094598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Frac-950 Tube Racks, Rack C</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">18-6083-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fraction Collector Frac-950</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">18-6083-00</td>
			<td>Includes kits and accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heated/Refrigerated Circulator&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:121px;">13271-102</td>
			<td>Model 1156D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heating Oven FD Series</td>
			<td style="width:191px;">Binder</td>
			<td style="width:121px;"></td>
			<td>Model FD 115</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">HiLoad 16/60 Superdex 200 pg</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">17-1069-01</td>
			<td style="width:239px;">Discontinued--Replacement Product: HiLoad 16/600 Superdex 200 pg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">J-26 XPI Avanti Centrifuge</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">393126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JA 25.50 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">363055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JLA 8.1 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">969329</td>
			<td>Includes 1 L polyporpylene bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JS 5.3 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">368690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Laminar Flow Hood</td>
			<td style="width:191px;">Themo Scientific</td>
			<td style="width:121px;">1849</td>
			<td style="width:239px;">Forma 1800 Series Clean Bench</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Microplate Reader</td>
			<td style="width:191px;">TECAN</td>
			<td style="width:121px;"></td>
			<td>infinite M200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Mini-PROTEAN Tetra Cell</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:121px;">165-8004</td>
			<td style="width:239px;">4-gel vertical electrophoresis system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mini-PROTEAN TGX Precast Gels</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:121px;">456-9036</td>
			<td style="width:239px;">Any kDa, 15-well comb</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ni-NTA Column</td>
			<td>Bio-Rad</td>
			<td>737-2512</td>
			<td>49 ml&#160;volume ECONO-Column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plasmid Miniprep Kit</td>
			<td style="width:191px;">OMEGA bio-tek</td>
			<td style="width:121px;">D6943-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">PowerPac HC Power Supply</td>
			<td>Bio-Rad</td>
			<td style="width:121px;">164-5052</td>
			<td>250 V, 3 A, 300 W</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Round Bottom Polypropylene Copolymer Tubes</td>
			<td style="width:191px;">VWR</td>
			<td style="width:121px;">3119-0050</td>
			<td>50 ml&#160;tubes for JA 25.50 rotor</td>
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			<td height="21" style="height:21px;width:299px;">Spin-X UF Concentrators</td>
			<td style="width:191px;">Corning</td>
			<td style="width:121px;">431488, 431483</td>
			<td>&#160;20 and 6 ml; MWCO: 10,000 Da</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Subcloning Service</td>
			<td style="width:191px;">GenScript USA Inc.</td>
			<td style="width:121px;"></td>
			<td>Protein Services</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ultrasonic Processor&#160;</td>
			<td style="width:191px;">Cole-Parmer</td>
			<td style="width:121px;">18910445A</td>
			<td>Model CV18</td>
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			<td height="21" style="height:21px;width:299px;">Vortex-Genie 2</td>
			<td style="width:191px;">Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model G560</td>
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expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration

Engenharia de proteínas recombinantes utilizou Escherichia coli (E. coli) para sistemas de expressão de quase quatro décadas, e hoje E. coli é ainda o organismo hospedeiro mais utilizado. A flexibilidade do sistema permite a adição de porções tais como uma etiqueta de biotina (para interacção com estreptavidina) e proteínas funcionais maiores, como a proteína fluorescente verde ou proteína vermelho cereja. Além disso, a integração de aminoácidos não naturais como quelantes de iões de metais, grupos funcionais reactivos, exclusivamente sondas espectroscópicas, e as moléculas que conferem modificações pós-traducionais permitiu uma melhor manipulação de propriedades de proteína e funcionalidades. Como resultado desta técnica cria proteínas de fusão personalizáveis ​​que oferecem utilidade significativa para vários campos de pesquisa. Mais especificamente, a sequência da proteína biotinylatable foi incorporado em muitas proteínas alvo, devido à interacção de alta afinidade entre biotina com a avidina e estreptavidina. Esta adição tem ajudado no aumento da detecção e purificação de proteínas marcadas, bem como abrindo o caminho para aplicações secundárias, tais como separação de células. Assim, as moléculas marcadas com biotina mostram uma influência crescente e generalizada nos campos Bioindustrial e biomédicas. Para efeitos de nossa pesquisa, temos projetado proteínas de fusão biotinilado recombinantes contendo fator de crescimento do nervo (NGF) e semaphorin3A (SEMA3A) regiões funcionais. Nós temos descrito anteriormente como estas proteínas de fusão biotinilado, juntamente com outras sequências de proteínas activas, pode ser preso a biomateriais para a engenharia de tecidos e efeitos regenerativos. Este protocolo descreve os conceitos básicos de proteínas biotinylatable engenharia na escala miligrama, utilizando um vetor lac induzível T7 e E. hospedeiros de expressão de E. coli, a partir da transformação de aumento de escala e purificação.

<html> Clonagem e Expressão de teste Quando chapeamento é realizado corretamente, único colônias isoladas devem formar a aumentar as chances de arrancar bactérias clonal transformaram células (Figura 2A). No entanto, se demasiadas células são plaqueadas, as placas são incubadas por muito tempo, a 37 ° C ou a transformação é questionável, as colónias podem cobrir a placa de ágar ou de formar agregados maiores de células (Figuras 2B</st...

Watch this Scientific Journal Video about Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration at JoVE.com

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

Desenvolvimento de proteínas de fusão biotinylatable tem muitas aplicações potenciais em diversas áreas de pesquisa. Engenharia de proteínas recombinantes é um procedimento para a frente que é eficaz em termos de custos, rendimentos elevados de proteínas personalizados.

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 ...

expression-isolation-purification-soluble-insoluble-biotinylated

Research

JoVE Journal

Bioengineering

33.4K visualizações.  University of Akron.  Desenvolvimento de proteínas de fusão biotinylatable tem muitas aplicações potenciais em diversas áreas de pesquisa. Engenharia de proteínas recombinantes é um procedimento para a frente que é eficaz em termos de custos, rendimentos elevados de proteínas personalizados. 

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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Uso de Células-tronco humanas perivasculares para Regeneração Óssea

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Bioengenharia

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sem contato, Monitoramento Label livre de células e matriz extracelular utilizando Espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Criando Gradientes adesivas e solúvel para a migração celular Imaging com Microscopia de Fluorescência

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Design e Construção de Matriz Extracelular Artificial (MECa) Proteínas de<em&gt; Escherichia coli</em&gt; Para engenharia de tecidos da pele

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Uma plataforma microfluídica para a isolação de alta taxa de transferência de célula única e Cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

Isolamento de Murino adiposo derivada de tecido microvascular Fragmentos como Vascularização Unidades para engenharia de tecidos

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Engenharia de Tecido Tridimensional de Redes de Astrócitos Alinhadas para Recapitular Mecanismos de Desenvolvimento e Facilitar a Regeneração do Sistema Nervoso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolamento de células-tronco mesenquimais do periósteo Alveolar humano e efeitos da vitamina D na atividade osteogênica do periósteo-derivado de células

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

Processo de moldagem baseados em molhado-fiação de gelatina para regeneração de tecidos

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...