Name: hela based cell free expression systems for expression of plasmodium rhoptry proteins
Uploaded: 2015-06-10T14:00:00
Description: Watch this Scientific Journal Video about HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins at JoVE.com

This research was supported by Cleveland State University Faculty Development Funds.

1.准备寄生虫细胞培养，收集寄生虫微丸<ol><li>加入10克的RPMI-1640粉末，66毫克硫酸庆大霉素的，2克碳酸氢钠，5.9克HEPES缓冲液，50毫克次黄嘌呤和3.8g的葡萄糖在1L制备的RPMI-1640-HEPES培养基的无菌卫生署2 O.混合使用表磁力搅拌器，直到1升蒸馏水中所有的粉末溶解。执行使用0.22微米的过滤介质微过滤。存储介质中的无菌瓶中。 </li><li>未感染的洗净（鲜）型使用RPMI A +红细胞。制备红细胞的5...

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使用两个步骤在体外人细胞表达系统和纯化蛋白的成功表达的重要步骤是：1）成功的扩增和基因引入pT7CFE-CHIS质粒载体的克隆; 2）转录的RNA在30℃;蛋白3）翻译在30℃在RNA酶抑制剂的存在; 4）开发使用涂覆有镍和5树脂珠）上免疫印迹分析的结果使用多克隆和单克隆抗体的亲和纯化方案。用于使用一个步骤的IVT偶联蛋白翻译系统蛋白质的成功表达的重要步骤是：1）成功的扩增和基因引入pT7CFE...

在疟疾研究，使用原核或真核细胞的系统免疫原性蛋白的表达仍然是一个挑战。 疟原虫的基因组的和未知的翻译后机制14,7，的AT丰富有助于与获得正确折叠的和免疫原性蛋白产生抗体和疫苗研究的困难。原核系统如大肠杆菌已被用于重组蛋白质表达。原核系统是低成本，有效，产生重组蛋白的高产率 并有多个克隆载体。 E.主机大肠杆菌细胞很容易变换和细胞生长迅速。然而，原核系统有例如缺乏氨基酸取代，翻译后修饰，污染，异构产品和重组蛋白的积累的包涵体24内危险的限制。 在一个研究由Mehlin 等人（2006年），1000的开放阅读框（ORF）表达。约7％的表达的蛋白质的可溶于14。所观察到的不溶性是由于基因的偏压性质和密码子的高频率理想地使用由疟原虫富含AT的基因组14。克服该问题的另一种策略已开发通过使用质粒或含有的tRNA识别稀有密码子或匹配频率3中的密码子的宿主细胞。即使在执行这些优化后，蛋白质的非常小的部分被表示为可溶的，活性和免疫原性14。无细胞表达系统包含了所有必需的转录和翻译元件，如核糖体，起始因子，延伸因子（翻译因子），tRNA和氨酰基tRNA合成酶。转录和翻译反应被耦合在一步法11,29。该transcription反应在一个试管中进行前mRNA的适当量的孵育翻译机器在不同的管11,29。虽然这些方法是成功的疟原虫属蛋白的表达，一个主要缺点是时间为蛋白质的表达，这是约22小时29的长度。此外，耗材列入成本高的协议29，细胞裂解液的劳动密集型筹备和不一致成分制剂，使这些系统无法实现的。研究人员对无细胞表达系统发展的主要焦点取决于多种因素，如快速的遗传修饰，具有较高的浓度和直线前进裂解液制剂1快产量。 真核系统例如酵母，哺乳动物细胞系，杆状病毒介导的表达系统， 四膜虫thermophilia，粘菌和寄生表达系统ス通道作为利什曼已经用于重组蛋白质表达7。真核系统中共享的亲缘关系，因此也共享特性，如糖基化，酰化，二硫键的形成，分子伴侣相互作用，蛋白水解和亚细胞区室。在真核生物中蛋白分泌事件预防的积累和降低毒性表达系统13。利用酵母系统是优选的，因为它们是适合于在大规模蛋白质表达和获得高的产率4。两个P.疟原虫蛋白PfCP-29和Pvs25用酵母系统4产生。然而，在大多数的蛋白质的合成的主要缺点是不规则N和O-糖基化模式，不当折叠和从它们的天然形式4蛋白的截断。 使用哺乳动物细胞合成重组蛋白质的是劳动密集型的并且它expen西伯来维持稳定的重组细胞系4。因此，哺乳动物细胞被限制为蛋白信号，蛋白质相互作用和寄生虫-宿主相互作用的分析，而且还用于测试DNA疫苗4。人类DNA和mRNA 体外蛋白表达的无细胞系统本文中所描述的HeLa细胞来源的哺乳动物为基础的系统表达的蛋白质中3小时。蛋白表达使用pT7CFE1-CHIS表达载体已C-末端标签促进鉴定和纯化。的HeLa细胞为基础的系统中补充有翻译起始因子和翻译调节器，从而提高翻译系统的效率。蛋白质可迅速转化，筛选，验证和在各种免疫学和结构应用15处理以供使用。 目前用于变种的表达的真核细胞表达系统，例如小麦胚芽，兔网织红细胞白条真核蛋白，包括恶性蛋白质11,29。另外，E。大肠杆菌基于无细胞系统也用于真核蛋白表达11。 表1通过比较系统的功能，以及易于使用的系统用于蛋白质表达的总结了在不同的分类细胞表达系统。相较于其他人无细胞系统必须执行后并共翻译修饰的能力，并且是不太昂贵。密码子优化是可能的，并且所有的反应可以在3小时来进行。的HeLa细胞系统是一种理想的翻译系统蛋白质合成在实验室设置。 在其它细胞系统人类自由细胞表达系统的一个主要优点是，从不同的器官和组织来源的多种不同的细胞系的可用性。几个品种无细胞系统可根据不同的细胞系进行设计。提取物从哺乳动物细胞衍生s具有更高的效率来合成大量的蛋白质，比任何其他的分类细胞表达系统。这些无细胞表达系统也可用于诊断和蛋白质表征的应用，如微阵列30。流行的HeLa细胞系是最广泛地用于进行蛋白33的表达。在HeLa细胞系内质网不发达，导致缺乏翻译后糖基化修饰的活性33。然而，该系统被认为是有利的疟原虫蛋白合成疟原虫也缺乏糖基化交翻译修改29。 HeLa细胞游离转录 - 翻​​译协议是容易执行的，廉价和蛋白质可表示在90分钟，准备用于纯化和进一步的下游应用。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Two-step in vitro human cell free expression system</td>
			<td>Thermo fischer</td>
			<td>88856</td>
			<td>Discontinued. Always store at -80oC. Do not thaw in warm water</td>
		</tr>
		<tr>
			<td>One-step in vitro coupled translation system</td>
			<td>Thermo fischer</td>
			<td>88860</td>
			<td>Always store at -80oC. Do not thaw in warm water</td>
		</tr>
		<tr>
			<td>ProBond Purification system</td>
			<td>Invitrogen</td>
			<td>K850-01</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Probond Nickel-Chelating Resin</td>
			<td>Invitrogen</td>
			<td>R801-01</td>
			<td>Store at 4oC.</td>
		</tr>
		<tr>
			<td>&#160;A+ Human Blood</td>
			<td>Interstate Blood Bank</td>
			<td></td>
			<td>Store at 4oC.</td>
		</tr>
	</tbody>
</table>

hela based cell free expression systems for expression of plasmodium rhoptry proteins

疟疾导致显著的全球发病率和死亡率。没有常规疫苗是当前可用。其中一个主要的原因，缺乏疫苗是确定合适的候选疫苗的挑战。疟疾蛋白使用原核和真核基于细胞的表达系统很差糖基化，通常不溶性且经受不当折叠导致降低的免疫原性表达。小麦胚芽，兔网织红细胞裂解物和大肠杆菌裂解物无细胞表达系统目前用于疟疾蛋白的表达。然而，表达时间和不适当的糖基化蛋白质的长度仍然是一项挑战。我们使用的HeLa基于无细胞表达系统，被称为“ 在体外人的分类细胞表达系统”表明在体外恶性疟原虫蛋白质的表达。 2.基于海拉细胞自由表达系统表达转录在75分钟和3微升transcrib的编的mRNA足以翻译的蛋白质在90分钟。的1步表达系统是一个转录和翻译偶联表达系统;转录和共翻译发生在3小时。该方法也可以通过提供额外的能量扩展6小时。在2步表达系统，表达被第一转录，然后添加至翻译组合用于蛋白质表达。我们描述了如何表达疟疾蛋白;疏水PF3D7_0114100毛雷尔的唇腭裂 - 2跨膜（PFMC-2TM）的蛋白质，亲水PF3D7_0925900蛋白质和含蛋白质PF3D7_1361800犰狳重复使用的HeLa细胞基于自由表达系统。的蛋白质表达于微体积采用2步和1-步骤表达的策略。亲和纯化方法纯化25μl的使用也被描述的体外人细胞表达系统表达的蛋白质。蛋白质产量是由布拉德福德测定法测定和表达和纯化的蛋白质可以通过蛋​​白质印迹分析来证实。表达的重组蛋白可用于免疫接种，免疫测定和蛋白质测序。

通过反应性的特异性抗体表达的重组蛋白的验证是在确认表达的蛋白质的正确折叠的重要的第一步。重组疟疾蛋白采用一步法和两步体外培养的人无细胞表达系统表达。重组蛋白是纯化的镍螯合亲和方法。然后，我们使用抗血清在SDS-PAGE分离的重组蛋白的Western印迹整个裂殖子棒状体。 图1示出PY17X_1139200，PY17X_1402200，PY17X_1366000，PY17X_0830000和PF3D7_1436300在1％琼脂糖?...

Watch this Scientific Journal Video about HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins at JoVE.com

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

基于细胞系统疟疾蛋白的表达仍然具有挑战性。我们证明两步并一步IVT（ 体外翻译）无细胞表达系统从HeLa细胞表达的重组疟疾蛋白质棒状体。我们使用的Ni树脂基于亲和性的纯化系统纯化棒状体蛋白质。

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

Research

JoVE Journal

Biology

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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

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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...