Name: a high yield and cost-efficient expression system of human granzymes in mammalian cells
Uploaded: 2015-06-10T15:00:00
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.生产表达质粒pHLsec-GZM的<ol><li>从人NK细胞制备总RNA用合适的RNA分离的方法和使用第一链cDNA合成试剂盒反转录（在26或NK细胞系的NK-92表达所有五个人类Gzms制备为原代细胞），继manufacturer`的建议。使用GZM基因PCR和克隆到pHLsec在23（拉杜Aricescu和伊冯·琼斯，英国牛津大学样的礼物）描述使用AGEI和KpnI位点（ 图1）放大。 注意：使用在表1所示的以下引物?...

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GzmA和GZMB的经典和广泛研究的作用是诱导凋亡在哺乳动物细胞中的其胞质递送由所述孔形成蛋白PFN 1之后。最近，Gzms的细胞毒性谱显著从哺乳动物细胞扩大到细菌9,10，以及某些寄生虫11。此外，GzmA和GZMB的非经典，胞外功能以及各种孤儿Gzms的生物学意义仍不清楚。因此，Gzms的健壮时间和成本的高效表达和纯化系统中，通过该协议规定，将是对这些未来的研究有很大的帮助...

所述Gzms是一族局部存在的CTL专门溶酶体和NK细胞1高度同源的丝氨酸蛋白酶。这些杀伤细胞的细胞毒性颗粒还包含与膜破坏蛋白质PFN和GNLY被在识别去往消除2,3靶细胞的同时释放与Gzms。有五个Gzms在人类（GzmA，B，H，K和M），和10 Gzms小鼠（GzmA - G，K，M和N）。 GzmA和GZMB是最丰富的和广泛的研究在人类和小鼠1。然而，最近的研究已经开始研究细胞死亡途径以及由在健康和疾病4，另外，所谓孤儿Gzms介导的额外的生物效应。 该Gzms最著名的功能，特别是GzmA和GZMB的，是程序性细胞死亡在哺乳动物细胞中的诱导时由PFN 5,6-递送到靶细胞。然而，最近的研究还PFN 7,8展示了Gzms细胞外效应与免疫调节和炎症产生深远的影响，独立胞浆交付。这是在Gzms胞浆入境后有效地杀伤细胞的光谱也于近日从哺乳动物细胞扩大至9,10细菌，甚至某些寄生虫11。最近的这些发现开辟了一个全新的领域进行研究GZM。因此，经济高效，高产哺乳动物表达系统将显著缓解的方式为那些未来的研究。 天然人，小鼠和大鼠Gzms已成功地从CTL和NK细胞线12-14的颗粒级分纯化。然而，在我们手中的这样的纯化技术的产率小于0.1毫克/升细胞培养物（未发表的观测和12）的范围内。此外，单个GZM由诺特的色谱分离而不污染řGzms和/或蛋白质，也存在于该颗粒是具有挑战性（未公布的数据和12,14）。重组Gzms产生于细菌15，酵母16，昆虫细胞17和甚至在哺乳动物细胞如HEK 293 18,19。仅在哺乳动物表达系统承担产生重组酶具有相同于天然细胞毒性蛋白质的翻译后修饰的潜力。翻译后修饰已牵涉与通过内吞作用的特异性摄取和蛋白酶的靶细胞20-22内的细胞内定位。因此，通过使用pHLsec 23质粒骨干GZM表达（拉杜Aricescu和伊冯·琼斯，英国牛津大学一个样的礼物），我们建立了一个简单，时间和成本效益的系统，高产蛋白生产HEK293T细胞。 pHLsec结合CMV增强用鸡Β肌动蛋白启动子;在一起，这些元件demonstra泰德在各种细胞系24中的最强的启动子活性。此外，该质粒含有兔Β珠蛋白内含子，优化的Kozak和分泌信号，一个赖氨酸的6xHis标签和一个多聚A信号。插入可方便地分泌信号，并确保对缺乏适当的N-末端结构域的蛋白的最佳表达和分泌效率的赖氨酸的6xHis标签（ 图1）之间进行克隆。为Gzms的表达，我们换成由载体随后肠激酶（EK）的网站（DDDDK）提供的分泌信号内源分泌信号序列，使得EK治疗活化分泌Gzms（活性Gzms开始与N-末端氨基酸序列IIGG 25）。另外，在有利于该方法，HEK293T细胞在低价介质快速增长，如Dulbecco改进的Eagle培养基（DMEM），并且非常适合于成本效益的磷酸钙转染法。

<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.

在下面的部分中，我们将提出一个GzmA制剂的一个完整的文件照亮方法。我们还成功纯化GZMB和GzmM，在生产方面的净化效率和活动类似的结果。然而，从这些准备工作后，我们将只显示几个选定的数据块。从293T细胞按照目前的协议GZMB准备在几个已发表 ​​的研究中使用，突出了他们活动的各种生物检测系统9,29,31-34。 一个典型的GzmA纯化示于图2A中 。有效?...

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.

人类颗粒酶在哺乳动物细胞中高产量和经济高效表达系统

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

Research

JoVE Journal

Biology

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Absolute Quantum Yield Measurement of Powder Samples

粉末样品的绝对量子产量测量

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Analysis of Cell Cycle Position in Mammalian Cells

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

哺乳动物链接组蛋白亚型表达分析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

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

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

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

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...