Name: overexpression and purification of human cis-prenyltransferase in escherichia coli
Uploaded: 2017-08-03T15:00:00
Description: Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

这项工作由以色列科学基金会结构细胞生物学研究中心（I-CORE）（1775/12）和以色列科学基金会资助，授予1721/16和2338/16（YH）和825/14（DK ）。对“田园遗产基金会”的支持表示高度赞赏。这项工作由Ilan Edri和Michal Goldenberg完成，部分履行了特拉维夫大学萨克勒医学院的MD论文要求。

1。克隆顺式 -PT在大肠杆菌中过表达 <ol><li>获得pET-32b表达载体，设计用于与109aa硫氧还蛋白（TRX）蛋白17融合的蛋白质序列的克隆和高水平表达，以及全长顺式 -PT的大肠杆菌密码子优化的18编码序列。 </li><li>照顾到具有TEV蛋白酶（烟草蚀纹病毒蛋白酶）切割位点（ENLYFQ / G，其中，“/”表示切割点）9，随后?...

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这里描述的用于纯化大肠杆菌细胞中功能性人DHDDS的方案是简单和有效的，允许一旦合适的构建体可用，在3-4天内过表达和纯化蛋白质。鉴于基因组测序的突破，蛋白质纯化的这些方案具有特殊的意义，这提供了许多关于许多疾病18遗传学的信息，因此需要开发高通量方法来表征蛋白质级19的致病机制。 为了克服异源蛋白过表达和纯?...

异戊二烯基转移是一组催化经由多个缩合反应1使用异戊烯二磷酸（IPP）2烯丙基二磷酸，的链延长酶。 Z型酶催化从缩合反应形成顺式双键，而E型酶催化反式双键形成3 。 顺式 -磷酸转移酶（ 顺式 -PT，Z型酶）根据其产物链长度经常分类为短链（C 15 ），中链（C 50-55 ）和长链（C 70-120 ） 4 。 DHDDS（脱氢二酰基二磷酸合成酶）是一种真核长链顺式 -PT，它通过与异戊烯基二磷酸（IPP） 1的多次缩合催化法呢基二磷酸（FPP，烯丙基二磷酸）的链延长，5，6。这导致脱氢二酰基二磷酸盐的形成，C 55-100多聚丙烯酰二磷酸酯作为焦磷酸二酰基焦磷酸酯的前体，参与N-连接蛋白糖基化的糖基载体分子1 。其中德系犹太人，在DHDDS导致常染色体隐性色素性视网膜炎7,8的错义非保守突变（K42E）。因此，本方案是为了获得适用于机械研究的纯化DHDDS而开发的。 大肠杆菌被认为是重要蛋白质表达最方便和最具成本效益的宿主，因此也是最常用的宿主。然而，当试图在大肠杆菌中异源过表达蛋白质时，应该考虑蛋白质特异性的考虑。获得正确的折叠，活动来自大肠杆菌的重组蛋白质不是由于不同蛋白质的不同特性而简单的。已经开发了许多方法来克服这些障碍。在这里，蛋白质融合的使用，优化的培养条件和密码子优化被用于允许功能活跃的人DHDDS在大肠杆菌中的过表达和纯化。值得注意的是，以前尝试过度表达酵母顺式 -PT而没有蛋白质融合，因为即使在洗涤剂12的存在下也完全不溶，因此不成功。所述方案简单，经济有效，节省时间，并允许获得适合机械研究的DHDDS制剂。鉴于不同物种中顺式 -PT的同源性，我们建议该方案也可以应用于其他真核的顺式 -PT。

<table width="751">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">pET-32b</td>
			<td style="width:100px;">Novagen</td>
			<td style="width:119px;">69016-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Express&#160;lysY&#160;Competent&#160;E. coli&#160;(High Efficiency)</td>
			<td style="width:100px;">NEB</td>
			<td>C3010I</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:365px;height:42px;">cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td style="width:100px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TALON-superflow resin</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28-9574-99</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiPrep 26/10 desalting column&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>17508701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiLoad 16/60 superdex-200&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">superdex-200 increase 5/150 GL&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28990945</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14C-Isopentenyl pyrophosphate</td>
			<td style="width:100px;">Perkin-Elmer</td>
			<td>NEC773050UC&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">trans,trans-Farnesyl pyrophosphate</td>
			<td style="width:100px;">Sigma</td>
			<td>44270-10MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

overexpression and purification of human cis-prenyltransferase in escherichia coli

丙二酰转移酶（PT）是一组通过多次缩合反应使用异戊烯基二磷酸酯（IPP）催化烯丙基二磷酸的链延长的酶。 DHDDS（脱氢二酰基二磷酸合成酶）是通过与异戊烯基二磷酸酯（IPP）的多次缩合催化法呢二磷酸（FPP，一种烯丙基二磷酸酯）催化链延长的真核长链顺式 -PT（从缩合反应形成顺式双键）。 DHDDS具有生物医学重要性，因为酶中的非保守突变（K42E）导致色素性视网膜炎 ，最终导致失明。因此，本协议是为了获得大量纯化的DHDDS而开发的，适用于机械研究。在这里，蛋白质融合的使用，优化的培养条件和密码子优化被用于允许功能活跃的人类DHDDS的过表达和纯化<e米&gt;电子。大肠杆菌描述的协议是简单，成本效益和时间节省。不同物种中顺式 -PT的同源性表明，该方案也可以应用于其他真核的顺式 -PT，如涉及天然橡胶合成的那些。

这里使用的构造和纯化过程的一般概述如图1所示。在每个纯化步骤获得的样品如图2所示。该SDS-PAGE分析显示DHDDS的逐步纯化，得到高度纯化的产物。 图3显示了纯化酶的分析SEC的结果，显示蛋白质仅被观察为同二聚体。 图4显示代表性的时间依赖性活性测定。 14 C-IPP掺入明显?...

Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

来自大肠杆菌的非变性条件下用于过表达和纯化密码子优化的人顺式 -丙烯基转移酶的简单方案 大肠杆菌 ，以及酶活性测定法。该方案可以推广用于生产适合于机械研究的数量和质量的其它顺式异戊烯基转移酶蛋白质。

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Research

JoVE Journal

Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

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在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...