Name: gst-his purification: a two-step affinity purification protocol yielding full-length purified proteins
Uploaded: 2013-10-29T17:50:00
Description: Watch this Scientific Journal Video about GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins at JoVE.com

我们感谢安妮 - 玛丽·迪翁 - 科特的讨论导致了该方法的开发。 JK和RB是FQNRT博士学位的学者，米-MG是凡尼尔CIHR学者，和J.-YM是一个FRSQ高级研究员。这项工作是由自然科学和工程研究理事会JY资金支持。 M。

1。生产重组杆状病毒的使用Invitrogen的BAC-到-BAC杆状病毒表达系统主要是根据制造商的协议，只有轻微的修改所产生的杆状病毒： <ol><li>修改后pFastBac1矢量（Gibco公司，生活）是创建一个包含GST和他-标签（ 图2）。一个的pET-52b中（+）载体（Novagen）中的多克隆位点（MCS），含有10xHis-标签，插入一个pFastBac1载体的MCS，生成pFastBac1 - 链霉素-10xHis。在PET-52B（+）的MCS?...

<ol>
	<li>Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J., Tan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+affinity+tags+for+protein+purification.">Comparison of affinity tags for protein purification.</a> Protein Expr. Purif. 41, 98-105 (2005).</li><li>Thain, A., Gaston, K., Jenkins, O., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+separation+of+GST+fusion+proteins+from+co-purifying+GroEL.">A method for the separation of GST fusion proteins from co-purifying GroEL.</a> Trends Genet. 12, 209-210 (1996).</li><li>Carr, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+recombinant+form+of+the+V+antigen+of+Yersinia+pestis,+using+three+different+expression+systems.">Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems.</a> Vaccine. 18, 153-159 (1999).</li><li>Armisen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adsorption+of+poly-His+tagged+glutaryl+acylase+on+tailor-made+metal+chelate+supports.">Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.</a> J. Chromatogr. A. 848, 61-70 (1999).</li><li>Kuhn, S., Zipfel, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculovirus+expression+vector+pBSV-8His+directs+secretion+of+histidine-tagged+proteins.">The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins.</a> Gene. 162, 225-229 (1995).</li><li>Buisson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+of+breast+cancer+proteins+PALB2+and+piccolo+BRCA2+in+stimulating+homologous+recombination.">Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.</a> Nat. Struct. Mol. Biol. 17, 1247-1254 (2010).</li><li>Filimonova, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoforms+of+Serratia+marcescens+nuclease.+The+role+of+Mg2++in+the+hydrolysis+mechanism.">Isoforms of Serratia marcescens nuclease. The role of Mg2+ in the hydrolysis mechanism.</a> Biochemistry. 62, 983-988 (1997).</li><li>Thorslund, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+breast+cancer+tumor+suppressor+BRCA2+promotes+the+specific+targeting+of+RAD51+to+single-stranded+DNA.">The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA.</a> Nat. Struct. Mol. Biol. 17, 1263-1265 (2010).</li><li>Genois, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+BRCA2+and+RAD51+for+promoting+homologous+recombination+in+Leishmania+infantum.">Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.</a> Nucleic Acids Res. 40, 6570-6584 (2012).</li><li>Shrestha, B., Smee, C., Gileadi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+vector+system:+an+emerging+host+for+high-throughput+eukaryotic+protein+expression.">Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression.</a> Methods Mol. Biol. 439, 269-289 (2008).</li></ol>

这里提出的GST-他的纯化方案，适用于范围广泛的重组蛋白大小的净化：成功纯化李 RAD51（41kDa），piBRCA2（120kDa），PALB2（130kDa），和一个不稳定的大分子量的蛋白质婴儿利什曼原虫 ： 李 BRCA2（125kDa）6,9。此外，与此协议纯化蛋白生化活性6。这种方法的成功完全取决于所需蛋白质的表达，溶解性和稳定性。 在其他宿主中的表达的分子生物?...

重组蛋白质的纯化是至关重要的，以解决在生物化学的基本问题。蛋白纯化的常规方法类似的离子交换层析和大小排阻层析依赖于目标蛋白的物理性质，例如它的等电点和电荷或大小，分别。后者蛋白质特性是由多种蛋白质，这增加了相当大的常规蛋白质纯化策略污染蛋白的机会共享。这个问题可能会被规避与使用多个纯化柱，这是耗时的。在同一时间，后者的色谱方法需要昂贵的实验装置。亲和标记纯化大力增加目标特异性，因为在大多数的情况下，标签将是唯一的目标蛋白。在最近的研究中，标志或HA-亲和纯化，得到了广泛的使用。 而相比之下，现有的拍摄其中单个标签用于ombinant蛋白质纯化的协议，我们建立两个标签的独特组合。我们的方法是融合GST标签的在N-末端和His-标签在感兴趣的蛋白质的C-末端，用于量和所需蛋白质的纯度之间的最佳比例。的GST是一个长标记（29 kDa）的，这是对纯化的谷胱甘肽琼脂糖珠高效。此外，使用GST保证我们的方法1的成本效益。裂解掉的GST与断前酶（具有识别序列LeuGluValLeuPheGln / GlyPro，导致增加了只有两个氨基酸）的可能性，具有许多优点，例如，这样的策略可避免的生理功能的蛋白质，由于变构阻改变。小的His-tag融合至其它蛋白质末端提供在第二纯化步骤中通过洗涤掉裂解的GST，以及降解的蛋白质和其他增加蛋白质纯度<html>污染物。此外，从GST切换到他的净化步骤柱（TALON树脂）时，协议不需要一个透析步骤。在这样的净化过程常见的污染物是热休克蛋白（HSP）。加入一种培养步骤含MgCl 2和ATP的允许移除这些污染物（ 图1）。 快速蛋白液相色谱（FPLC）和高效液相色谱法（HPLC）是依赖于昂贵的仪器来产生高收益感兴趣的纯化蛋白质的常用技术。我们在座的批次纯化方案，相反，是手动的，不需要昂贵的仪器设置。高产量的蛋白质可以通过扩大的协议达成。在同一时间，用批量纯化方案中，洗脱体积可以提高蛋白质浓度，这是不符合FPLC或HPLC给予调整。 ntent“&gt;此外，在批次GST-他的纯化方案，一个大小范围的〜10-300 kDa的蛋白可被纯化。其中最大的优点是通过以下事实给出，人们可以同时净化几种蛋白质举例来说，两个野生型及其突变体蛋白。所提出的协议的成功完全依赖于目的蛋白质的表达水平和溶解度。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>E.Coli DH10Bac (competent)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus Expression System</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Maxi Prep Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Solution I and II</td>
		</tr>
		<tr>
			<td>Ultra Pure Bluo-Gal</td>
			<td>Gibco (Life)</td>
			<td>15519028</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gibco (Life)</td>
			<td>15529019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 infected cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>PreScission enzyme</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;s infected medium supplemented</td>
			<td>Gibco (Life)</td>
			<td>11605-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum characterized</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>P/S (Penicillin-Streptomycin)</td>
			<td>Gibco (Life)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose resin</td>
			<td>GE bioscience</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Talon resin</td>
			<td>Clontech</td>
			<td>635504</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>BioShop</td>
			<td>288324</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco (Life)</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal GST-antibody</td>
			<td>Production in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 6X-His-antibody</td>
			<td>Clontech</td>
			<td>631212</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dounce homogenizer (tight)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator (Fisher dismembrator)</td>
			<td>Fisher</td>
			<td>Model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (50 mm)</td>
			<td>Fisher Scientific</td>
			<td>2115217</td>
			<td></td>
		</tr>
	</tbody>
</table>

gst-his purification: a two-step affinity purification protocol yielding full-length purified proteins

在酶学测定法的关键蛋白质的体外生化特性的必要的高浓度的感兴趣的纯化蛋白。蛋白质纯化方案应结合效率，简单性和成本效益1。在这里，我们描述了GST-他的方法作为一种新的小型的亲和纯化系统的重组蛋白，根据N-末端谷胱甘肽琼脂糖标签（GST）的2,3和C-末端10xHis标签4，它们都稠合到感兴趣的蛋白质。后者构建体被用于产生杆状病毒的Sf9对感染细胞中的蛋白质表达5的感染。消费税是用来确保净化效率相当长的标记（29 kDa的）。然而，它可能影响到蛋白质的生理特性。因此，它使用的是断前酶6随后裂解的蛋白。为了确保最大的纯度和去除切割的商品及服务税，我们新增的基础上，比较小His标签的第二亲和纯化步骤。重要的是，我们的技术是基于两个不同的标记侧翼的蛋白质，这是一个有效的工具来除去降解的蛋白质，因此两端，丰富了全长蛋白。这里提出的方法不需要昂贵的仪器设置，诸如FPLC。此外，我们成立氯化镁和ATP清洗以去除热休克蛋白杂质和核酸酶处理，取消污染核酸。总之，两个不同的标记侧翼的组合的N-和C-末端和切割过的标签1的功能，担保感兴趣的高度纯化的和全长蛋白质的回收率。

为了说明GST-HIS纯化方案的效率，我们纯化Rec14，一个S。 32.9 kDa的酵母蛋白质。该Rec14 cDNA克隆在我们修改pFastBac1向量允许GST和他-标签的添加在N-和C-末端，分别为（ 图1A）。重组杆状病毒，然后准备并用于感染SF9感染的细胞用于蛋白质表达。可溶性细胞裂解物与GST珠和束缚蛋白裂解与断前蛋白酶的消费税洗脱。所得Rec14-His蛋白是亲和纯化TALON树脂和束缚的蛋白质用含有500mM...

Watch this Scientific Journal Video about GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins at JoVE.com

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

在本协议中，我们展示了一个高效和具有成本效益的小型蛋白质纯化方法，它允许重组蛋白的纯化结合独特的可裂解GST标签和一个小His标签。

GST-他的净化：一个两步亲和纯化协议屈服全长纯化的蛋白质

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Research

JoVE Journal

Biology

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 ...

科研

生物学

Purification of Hsp104, a Protein Disaggregase

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

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 ...

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 ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

蛋白质纯化方法测定结合亲和力微尺度热泳

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...