Name: a high yield and cost-efficient expression system of human granzymes in mammalian cells
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Description: Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

1. Produção do plasmídeo de expressão pHLsec-GZM <ol><li> Preparar ARN total a partir de células NK humanas (células primárias preparadas como no 26 ou a linha celular NK NK-92 expressando os cinco Gzms humanos) utilizando um método de isolamento de ARN adequada e transcrever reversa usando um kit de ADNc de primeira cadeia a sintetizar seguintes manufacturer` s recomendações. GZM amplificar ADNc utilizando PCR e o clone em pHLsec como descrito em 23 (presente tipo de Radu Aricescu e Yv...

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O papel clássico e extensivamente estudada de GzmA e GzmB é a indução de apoptose em células de mamífero após a sua entrega por citosólico da proteína formadora de poros PFN 1. Recentemente, o espectro citotóxica dos Gzms foi alargado significativamente a partir de células de mamíferos para bactérias 9,10, bem como para certos parasitas 11. Além disso, as funções não clássicos, extracelulares de GzmA e GzmB, bem como a significância biológica dos vários Gzms órfãos...

Os Gzms são uma família de serina-proteases altamente homólogas localizadas nos lisossomas especializados de CTL e as células NK 1. Os grânulos citotóxicos destas células assassinas, também contêm as proteínas de membrana de desregulação e PFN GNLY que são libertadas em simultâneo com as Gzms no reconhecimento de uma célula alvo destinado à eliminação 2,3. Existem cinco Gzms em seres humanos (GzmA, B, H, K e M), e 10 em ratos (Gzms GzmA - G, K, M e N). GzmA e GzmB são os mais abundantes e extensivamente estudados em humanos e ratos 1. No entanto, estudos mais recentes começaram a investigar os percursos de morte celular, bem como os efeitos biológicos adicionais mediadas pelos outros, os chamados Gzms órfãos na saúde e na doença 4. A função mais conhecida das Gzms, em particular de GzmA e GzmB, é a indução de morte celular programada em células de mamífero quando entregue nas células alvo por PFN 5,6. Porém, Estudos mais recentes também demonstraram efeitos extracelulares dos Gzms com profundo impacto sobre a regulação imune e inflamação, independentemente da entrega cytosolic pela PFN 7,8. O espectro de células que são mortos de forma eficiente após a entrada cytosolic dos Gzms também foi recentemente alargado a partir de células de mamíferos para 9,10 bactérias e até mesmo certos parasitas 11. Estas descobertas recentes abriu um novo campo para pesquisadores GZM. Portanto, um, de alto rendimento do sistema de expressão de mamífero custo-eficiente irá facilitar significativamente o caminho para esses estudos futuros. Humanos, ratos e de camundongos Gzms nativas foram purificados com sucesso a partir da fracção de grânulos de CTL e células NK linhas 12-14. No entanto, nas nossas mãos o rendimento de tais técnicas de purificação está na gama de menos do que 0,1 mg / L de cultura de células (observação não publicada e 12). Além disso, a resolução cromatograf ica de um único GZM sem contaminação por outrr Gzms e / ou proteínas que também estão presentes nos grânulos é um desafio (dados não publicados) e 12,14. Gzms recombinantes foram produzidas em bactérias, leveduras 15 16 17, células de insecto e em ainda em células de mamífero, tais como células HEK 293 18,19. Apenas os sistemas de expressão de mamífero suportar o potencial para produzir enzimas recombinantes com modificações pós-tradução idêntica à proteína nativa citotóxico. Modificações pós-tradução têm sido implicados com a captação especifica por endocitose e a localização intracelular da protease dentro das células alvo 20-22. Portanto, usando pHLsec 23 (um presente tipo de Radu Aricescu e Yvonne Jones, da Universidade de Oxford, Reino Unido) como a espinha dorsal do plasmídeo para a expressão GZM, foi estabelecido um sistema simples, tempo e custo-eficiente para a produção de proteínas de alto rendimento em HEK293T células. pHLsec combina um intensificador de CMV com um frango Β-actina promotor; juntos, esses elementos demonstrated a actividade do promotor mais forte em várias linhas de células 24. Além disso, o plasmídeo contém um intrão de coelho Β-globina, os sinais de secreção e de Kozak optimizada, um-6xHis-tag Lys e um sinal poli-A. As inserções podem ser convenientemente clonados entre o sinal de secreção e a-6xHis-tag Lys (Figura 1) assegurar a expressão óptima e eficiência de secreção para proteínas que não possuem domínios N-terminais adequados. Para a expressão das Gzms, que substituiu a sequência de sinal de secreção endógena com o sinal de secreção fornecida pelo vector, seguido por uma enteroquinase (EK) local (DDDDK), de modo que o tratamento EK activou a Gzms secretadas (Gzms activas começar com o N-terminal sequência de aminoácidos IIGG 25). Além disso, em favor deste método, as células HEK293T crescer rapidamente em baixa preço-médio, como meio de Eagle modificado por Dulbecco (DMEM), e são adequados para o método de transfecção com fosfato de cálcio eficiente em termos de custo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

Na seção seguinte, vamos apresentar uma documentação completa de uma preparação GzmA para iluminar o método. Nós também purificado com sucesso GzmB e GzmM, produzindo resultados semelhantes em relação à eficiência e atividade purificação. No entanto, a partir dessas preparações posteriores só vamos mostrar algumas peças seleccionadas de dados. Preparações GzmB de células 293T seguintes actual protocolo foram utilizados em vários estudos publicados, com destaque para a sua actividade em vários sis...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

A High Yield e custo-eficiente sistema de expressão de Granzimas Humanos em células de mamíferos

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

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