Name: one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)
Uploaded: 2014-04-20T14:00:00
Description: Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

我们非常感谢技术支持四逆Miettinen，明娜Pöllänen和塔如Rautavesi的。我们感谢赫尔卡Nurkkala提供的HEK 293细胞中表达的双链球菌标记的GFP和佩卡Evijärvi提供录音设备。这项工作是由芬兰科学院，授权号码138329，134684和258978。

1，交联链球菌-Tactin聚甲基丙烯酸酯树脂与双（磺基琥珀酰亚胺）的辛二酸（BS3） <ol><li>平衡含有2mg BS3交联剂的室温1密封微管中。注意：BS3是一种有害物质。戴防护手套和护目镜。 </li><li>重悬链球菌-Tactin聚甲基丙烯酸酯树脂（在100毫米的Tris-HCl，pH 8.0的50％悬浮液，1 mM的EDTA，150 mM氯化钠）进行了简短的剧烈震荡，并立即用枪头与600微升悬浮液转移到离心柱结束切断。 </li><li>离心机在1500×g离?...

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上述协议可用于纯化目的和其相关的复合物中的任何合适的缓冲液不含有生物素或强变性剂的任何双链球菌标签的诱饵蛋白。在该协议的当前版本中，结合和洗涤都相对严格的条件下在高盐和非离子型去污剂的存在下进行。尽管这会导致更少的背景，脆弱的蛋白质复合物可以解离这些条件下。为了保持这种低亲和力复合物，盐的浓度可被降低和非离子型洗涤剂，可减少或省略的结合和洗涤缓冲液?...

近年来，Strep-标签技术已成为广泛使用的生物医学研究中的许多领域，包括蛋白质组学和结构生物学。这种蛋白质纯化技术，这依赖于重组蛋白的融合到一个短的Strep-标签肽，已经成熟账面链球菌-Tactin，链霉的具有改进的肽结合的能力的基因工程变体亲和基质的到来。1，2含有两个拷贝Strep-标签II的指定双Strep-标签或SIII-标签，表现出对链球菌-Tactin基质比那些仅含有一个单一的Strep-标签，确保了重组蛋白质的更有效的纯化具有更高的亲和力和融合蛋白其相关的具有约束力的合作伙伴。然而，双链球菌标签蛋白对链球菌-Tactin较高的亲和力也有其不利的一面。这种蛋白质与过量的生物素的竞争性洗脱可能不完整，导致减少靶蛋白的产量。 A M矿石有效的替代方案是洗脱SDS，但它会导致与链球菌-Tactin不需要的样品污染从树脂上释放，使得用蛋白质组学分析测定不相容。本文提出了一种技术，通过第一稳定链球菌-Tactin的通过化学交联的树脂偶联的四聚体，然后用SDS洗脱从所得的交联树脂的双链球菌标记的蛋白质和其相关的复合物来克服这种限制。因此，足够的蛋白质产量可在不与链球菌-Tactin样品污染来实现，从而允许通过质谱法进一步分析。 该方法适用于任何重组融合蛋白的纯化与表面暴露SIII-标签3或双-Strep-标签（氨基酸序列WSHPQFEK（GGGS）3 WSHPQFEK和SAWSHPQFEK（GGGS）2 GGSAWSHPQFEK，分别）。该蛋白质可以是动物，植物或细菌来源的，可以从任一总细胞中分离裂解物或富集的细胞器部分。作为一个例子，我们在这里描述一个SIII标签蛋白VPG-Pro的马铃薯A病毒（PVA）4从PVA-感染的烟草本塞姆氏烟草植物的细胞核部分的纯化。如前所述5，具有以下修饰的核成分进 ​​行分离：细胞没有用甲醛处理，丁酸钠代替在所有的缓冲区用5mM氟化钠，完全蛋白酶抑制剂被置换为PMSF，加入Triton X-100的浓度在萃取缓冲器＃2降低到0.3％（体积/体积），并通过蔗糖垫离心得到（提取缓冲液＃3）的核沉淀再悬浮于1.45毫升预冷的结合缓冲液中并旋转1.5小时，在4℃下根据下面描述（见第2部分）的协议包含SIII标签的诱饵蛋白和相关的复合物（诱饵蛋白样品）所得的核提取物进行了处理。

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr>
			<td>Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2mg</td>
			<td>Pierce/Thermo Scientific</td>
			<td>21585</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.</a></td>
		</tr>
		<tr>
			<td>Strep-Tactin MacroPrep resin (50% suspension)</td>
			<td>IBA</td>
			<td>2-1505</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 &#956;m, non-sterile</td>
			<td>Costar (Corning)</td>
			<td>8163</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Dolphin-nose tubes</td>
			<td>Costar (Corning)</td>
			<td>3213</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Avidin&#160;</td>
			<td>IBA</td>
			<td>2-0204</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
	</tbody>
</table>

one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)

链球菌标记的融合蛋白对携带一个精心设计的链亲和素（链霉素-Tactin）树脂亲和纯化已成为生理条件下广泛使用的方法为蛋白质复合物的分离。含有两个拷贝Strep-标签II的指定双Strep-标签或SIII-标签，融合蛋白具有对链球菌-Tactin相比，那些仅含有一个单一的Strep-标签更高亲和力的优点，因而允许更有效的纯化。然而，这一优点是通过一个事实，即双链球菌标记的蛋白与生物素的洗脱可能不完整，导致低蛋白恢复抵消。回收可以通过使用变性洗脱剂十二烷基硫酸钠（SDS）得到显着改善，但是这会导致样品污染链球菌-Tactin从树脂上释放，使得与下游蛋白质组分析测定不相容。为了克服这种限制，我们已经开发出一种方法，其中树脂耦合链球菌-Tactin的四聚体首先，通过共价交联与双（硫代琥珀酰亚胺基）辛二酸酯（BS3），将所得的交联树脂，然后用纯化靶蛋白复合物在单一批次纯化步骤稳定化。高效洗脱SDS确保了良好的蛋白质回收，而没有污染链球菌-Tactin允许通过质谱下游蛋白分析。作为概念验证，我们在这里描述的协议为SIII标记病毒蛋白VPG-PRO从病毒感染N的核提纯使用链球菌-Tactin聚甲基丙烯酸酯树脂的交联与BS3 本塞姆氏烟草植物。相同的协议，可用于净化任何感兴趣的双链球菌标记的蛋白质和其生理结合配偶体进行表征。

纯化过程在图1中示意性地示出，以与其他现有的纯化方法相关联的问题的表示一起。 <img alt="图1" fo:content-width="5.5in" src="/files/ftp_upload/51536/51536fig1highres.jpg" width="650" /> 图1的纯化过程的示意图。的工作流程包括通过共价交联与BS3，双-链霉素标记的诱饵蛋白和相关的复合物结合到交联的树脂的树脂偶联链球菌-Tactin四聚?...

Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

对链球菌-Tactin树脂交联的双（磺基）辛二酸酯（BS3）双链球菌标记蛋白及其复合物的一步纯化

的方法被描述为在链亲和素修饰的双链球菌标记的融合蛋白和它们的特定配合物的净化效率（链球菌-Tactin）树脂共价交联的双（硫代琥珀酰亚胺基）辛二酸酯（BS3）。该方法具有速度快，良好的靶蛋白回收率和纯度高的优点，并且与随后的分析通过质谱兼容。

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

Research

JoVE Journal

Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

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 ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

一步代谢：碳水化合物，在一个单一的过程量化的有机和氨基酸

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

制备，纯化和磁共振成像造影剂的使用稀土配合物的表征

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

冷冻干燥细菌及其影响细胞生存的配方

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Purification of Transcripts and Metabolites from Drosophila Heads

Purification of Transcripts and Metabolites from Drosophila Heads

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...