Name: preparation of the mgm101 recombination protein by mbp-based tagging strategy
Uploaded: 2013-06-25T14:00:00
Description: Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

我们感谢斯蒂芬·威尔肯斯在透射电子显微镜的帮助。这项工作是支持由国家机构卫生部授予R01AG023731的。

以前的研究已经表明，第一个氨基末端的22个氨基酸残基的Mgm101裂解后，导入到线粒体19。对于在大肠杆菌中的表达，通过PCR扩增MGM101缺乏的前22个密码子的开放读码框放在男性的编码序列的表达载体pMAL-C2E的修改版本，麦芽糖结合蛋白（MBP）的下游。这会产生的MBP-Mgm101的的断前蛋白酶（ 图3）与一个连接器包含一个切割位点的融合。首次构建质粒选择E?...

<ol>
	<li>San Filippo, J., Sung, P., Klein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+eukaryotic+homologous+recombination.">Mechanism of eukaryotic homologous recombination.</a> Annu. Rev. Biochem. 77, 229-257 (2008).</li><li>Bishop, D. K., Park, D., Xu, L., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMC1:+a+meiosis-specific+yeast+homolog+of+E.+coli+recA+required+for+recombination,+synaptonemal+complex+formation,+and+cell+cycle+progression.">DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression.</a> Cell. 69, 439-456 (1992).</li><li>Shinohara, A., Ogawa, H., Ogawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad51+protein+involved+in+repair+and+recombination+in+S.+cerevisiae+is+a+RecA-like+protein.">Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.</a> Cell. 69, 457-470 (1992).</li><li>Passy, S. I., 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=Human+Dmc1+protein+binds+DNA+as+an+octameric+ring.">Human Dmc1 protein binds DNA as an octameric ring.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 10684-10688 (1999).</li><li>Story, R. M., Weber, I. T., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+E.+coli+recA+protein+monomer+and+polymer.">The structure of the E. coli recA protein monomer and polymer.</a> Nature. 355, 318-325 (1992).</li><li>Yu, X., Jacobs, S. A., West, S. C., Ogawa, T., Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+structure+and+dynamics+in+the+helical+filaments+formed+by+RecA+and+Rad51+on+DNA.">Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 8419-8424 (2001).</li><li>Conway, A. B., 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=Crystal+structure+of+a+Rad51+filament.">Crystal structure of a Rad51 filament.</a> Nat. Struct. Mol. Biol. 11, 791-796 (2004).</li><li>Bai, Y., Davis, A. P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+allele+of+RAD52+that+causes+severe+DNA+repair+and+recombination+deficiencies+only+in+the+absence+of+RAD51+or+RAD59.">A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.</a> Genetics. 153, 1117-1130 (1999).</li><li>Bai, Y., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rad52+homolog+is+required+for+RAD51-independent+mitotic+recombination+in+Saccharomyces+cerevisiae.">A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae.</a> Genes Dev. 10, 2025-2037 (1996).</li><li>Paques, F., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+of+recombination+induced+by+double-strand+breaks+in+Saccharomyces+cerevisiae.">Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.</a> Microbiol. Mol. Biol. Rev. 63, 349-404 (1999).</li><li>Lopes, A., Amarir-Bouhram, J., Faure, G., Petit, M. A., Guerois, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+novel+recombinases+in+bacteriophage+genomes+unveils+Rad52,+Rad51+and+Gp2.5+remote+homologs.">Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs.</a> Nucleic Acids Res. 38, 3952-3962 (2010).</li><li>Poteete, A. R., Sauer, R. T., Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+structure+and+quaternary+organization+of+the+bacteriophage+P22+Erf+protein.">Domain structure and quaternary organization of the bacteriophage P22 Erf protein.</a> J. Mol. Biol. 171, 401-418 (1983).</li><li>Passy, S. I., Yu, X., Li, Z., Radding, C. M., Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rings+and+filaments+of+beta+protein+from+bacteriophage+lambda+suggest+a+superfamily+of+recombination+proteins.">Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 4279-4284 (1999).</li><li>Ploquin, 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=Functional+and+structural+basis+for+a+bacteriophage+homolog+of+human+RAD52.">Functional and structural basis for a bacteriophage homolog of human RAD52.</a> Curr. Biol. 18, 1142-1146 (2008).</li><li>Chen, X. J., Guan, M. X., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGM101,+a+nuclear+gene+involved+in+maintenance+of+the+mitochondrial+genome+in+Saccharomyces+cerevisiae.">MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae.</a> Nucl. Acids Res. 21, 3473-3477 (1993).</li><li>Meeusen, 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=Mgm101p+is+a+novel+component+of+the+mitochondrial+nucleoid+that+binds+DNA+and+is+required+for+the+repair+of+oxidatively+damaged+mitochondrial+DNA.">Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA.</a> J. Cell Biol. 145, 291-304 (1999).</li><li>Mbantenkhu, 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=Mgm101+is+a+Rad52-related+protein+required+for+mitochondrial+DNA+recombination.">Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.</a> J. Biol. Chem. 286, 42360-42370 (2011).</li><li>Nardozzi, J. D., Wang, X., Mbantenkhu, M., Wilkens, S., Chen, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+properly+configured+ring+structure+is+critical+for+the+function+of+the+mitochondrial+DNA+recombination+protein.">A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein.</a> Mgm101. J. Biol. Chem. 287, 37259-37268 (2012).</li><li>Zuo, X., Xue, D., Li, N., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+core+of+the+mitochondrial+genome+maintenance+protein+Mgm101p+in+Saccharomyces+cerevisiae+determined+with+a+temperature-conditional+allele.">A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele.</a> FEMS Yeast Res. 7, 131-140 (2007).</li><li>Itoh, K., 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=DNA+packaging+proteins+Glom+and+Glom2+coordinately+organize+the+mitochondrial+nucleoid+of+Physarum+polycephalum.">DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum.</a> Mitochondrion. 11, 575-586 (2011).</li><li>Janicka, 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=A+RAD52-like+single-stranded+DNA+binding+protein+affects+mitochondrial+DNA+repair+by+recombination.">A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination.</a> Plant J. 72, 423-435 (2012).</li></ol>

它一直是一个挑战，产生一个稳定的，原生的重组Mgm101蛋白E.大肠杆菌可能是由于其在细菌细胞中的不溶性。在这份报告中，我们表明，MBP融合战略使蛋白质表示在相当高的水平。通过负染色透射电子显微镜和尺寸排阻色谱法，我们以前曾表明，MBP的融合蛋白在体外 18形成均匀的低聚物。这是可能的折叠和低聚Mgm101的，可能比较慢线粒体基质相比，细菌细胞中。 unoligomerized Mg...

同源重组是非常重要的双链断裂的修复（DNA双链断裂）和链间交联，并从倒塌的复制叉1重新开始DNA复制。在常规的同源重组，中央的反应由ATP-依赖的重组酶RecA蛋白在原核细胞中，在真核生物中1-3的Rad51和Dmc1。这些重组酶形成核蛋白丝的单链DNA，这是必不可少的双链DNA模板（ 图1，左侧面板）4-7内发起同源搜索和钢绞线入侵。除了 ​​常规的计划，同源重组也可以采取地方在RecA/Rad51-independent的方式（ 图1，右图）。例如，酵母的保守和Rad59蛋白质可以直接催化切除双链DNA断裂露出的互补单链DNA链退火。这个重组过程中，被称为唱乐链退火，一般不涉及同源配对的双链DNA模板。退火后，除去核酸外切酶的异源尾巴刻痕结扎以恢复基因组连续性8-10。常伴有直接重复区域之间的基因组序列缺失修复由单链退火机制。 RAD52属于普遍噬菌体11的重组蛋白质，是一个多元化的群体。这些蛋白质也被称为单链退火蛋白（会计实务准则），根据他们的活动在促进同源的单链DNA分子退火。最好噬菌体会计实务准则Redβ的的ERF从噬菌体λ噬菌体rac和葡激酶蛋白从噬菌体lactococcal UL36和P22，RECT。的会计准则结构其特征在于由一个典型的β-β-β-α倍，虽然相似，几乎检测不到能够在他们的主要序列。他们都形成大的同源寡聚体环10 - 14倍的对称性在体外 12-14。这一特点高阶的组织结构功能的影响还不是很清楚。 我们有兴趣了解线粒体基因组同源重组的机制。我们先前已经确定MGM101是必不可少的维护在酿酒酵母 15线粒体DNA的基因。MGM101后来发现线粒体拟核相关需要的耐受性线粒体DNA损伤剂16。然而，这项研究已Mgm101忍住在过去十年中的难度生产重组Mgm101。最近，我们已经成功地大量生产可溶性Mgm101在从E大肠杆菌使用的MBP融合策略。这使我们能够证明Mgm101股生化和结构的相似性与RAD52家族蛋白17,18。在这份报告中，三个步骤的纯化过程进行了描述，产生均匀Mgm101for的生化和结构分析（ 图2）。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Expression vector pMAL-c2E</td>
			<td>New England Biolabs</td>
			<td>#N8066S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PreScission Protease</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#27-0843-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BL21-CodonPlus(DE3)-RIL cells</td>
			<td>Strategene</td>
			<td>#230245</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Roche Applied Science</td>
			<td>#11034626001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Roche Applied Science</td>
			<td>#11359053001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Roche Applied Science</td>
			<td>#10837091001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>#DN25-1G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Roche Applied Science</td>
			<td>#11411446001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England Biolabs</td>
			<td>#E8021L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Econo-Column chromatography column</td>
			<td>BIO-RAD</td>
			<td>#7372512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td>
			<td>BIO-RAD</td>
			<td>#732-4130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS15RH02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 prep grade column</td>
			<td>Amersham Bioscirnces</td>
			<td>#17-0489-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS0611</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

20年前被确定MGM101基因维护线粒体DNA中的作用。从几组研究表明，的Mgm101蛋白参与线粒体DNA的重组修复。最近的调查表明，Mgm101 RAD52型重组蛋白家族有关。这些蛋白质形成大的低聚环促进退火同源单链DNA分子。然而，表征阻碍了Mgm101的生产重组蛋白的难度。在这里，用于制备重组Mgm101的一个可靠的程序进行说明。麦芽糖结合蛋白（MBP）标签Mgm101的首先在大肠杆菌中表达。最初是由直链淀粉的亲和层析柱纯化的融合蛋白。在被释放后通过蛋白水解裂解，Mgm101从MBP分离，通过阳离子交换色谱法。然后单分散Mgm101是获得通过尺寸排阻色谱法。可以定期获得的产量为〜0.87毫克每升细菌培养Mgm101。该的重组Mgm101有DNA污染最小。对样品成功地用于生化，结构和单粒子图像分析Mgm101。此步骤也可用于制备其它大的低聚物的DNA结合蛋白，可能会被错误折叠的和细菌细胞毒性的。

Mgm101是一个RAD52在线粒体相关的重组蛋白。 RAD52已被广泛研究，它的作用在线粒体DNA重组（ 图1）。重组Mgm101可以准备通过三个步骤（ 图2）。这是促进所使用的MBP的标记策略，允许以可溶形式表达在Mgm101从标签的蛋白水解裂解，随后被释放（ 图3）。 在一个典型的制备方法中，约2-3毫克，MBP-Mgm101融合可以被回收后，直链淀粉的亲和层析?...

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

酵母线粒体类核蛋白质，Mgm101，是一种重组蛋白RAD52型，形成大低聚环。甲协议的描述以制备可溶性的重组Mgm101使用麦芽糖结合蛋白（MBP）标记加上阳离子交换和尺寸排阻色谱法策略。

MBP基于标记策略的Mgm101重组蛋白的制备

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Research

JoVE Journal

Biology

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

利用数学建模预测的人口更替策略的有效性

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

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 GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

基因改造和重组唾液的腺体器官培养

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

提高蛋白质结晶的成功率随机Microseed矩阵筛选

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

分段微管的制备研究运动驱动的微管拆解完

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans