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。大肠杆菌转化，而 ​​不IPTG和的Xgal蓝色/白选择。将得到的质粒pMAL-C2E MGM101然后导入大肠杆菌大肠杆菌菌株BL21-CodonPlus（DE3）-RIL，选择氨苄青霉素和氯霉素抗性菌落。 1。表达，诱导，细胞裂解和DNase I处理<ol><li>伊诺克ulate新鲜的转化体在10ml补充有葡萄糖（0.2％），氨苄青霉素（100微克/毫升）和氯霉素（50微克/毫升）的LB培养基（1％蛋白胨，0.5％酵母提取物，1％氯化钠）。在200转摇孵育在37°CO / N。 </li><li>为2升与上述补充的LB培养基中，接种10毫升的预培养物。成长的细胞在37℃振荡，直到OD 600达到0.5。 </li><li>通过加入异丙基β-D-1-硫代吡喃半乳糖苷（IPTG），使最终浓度为0.3 mM，的MBP-Mgm101的融合蛋白的诱导表达。 5小时，在30℃振荡，细胞生长。 </li><li>收集细胞，通过离心分离，用Beckman JA-10转子（5500×g离心，4℃下，10分钟）。后弃去上清，重悬细胞沉淀在30ml含有蛋白酶抑制剂亮抑蛋白酶肽（25μM，1μM的胃酶抑素A和1mM的PMSF）的裂解缓冲液（的的20mM Tris-HCl，pH值7.4和1mM EDTA，pH值7.4）。 </li><li>超声细胞悬浮液，冰浴20秒使用超声波处理器（热系统，型号W-385），在占空比为50％时，允许在冰上冷却30秒，然后重复2倍。 </li><li>添加1毫升DNA酶1股票在2毫克/毫升。细胞裂解液在4℃下摇动2小时。 </li><li>调整的NaCl至终浓度为500 mM。 </li><li>中取出的细胞碎片。在4℃下以10,000 xg离心30分钟。 </li></ol> 2。与直链淀粉亲和层析纯化<ol><li>平衡1.5毫升的裂解缓冲液中的直链淀粉树脂（50％浆液）。平衡直链淀粉树脂加入细胞裂解物。轻轻摇动4°CO / N </li><li>加载到Econo柱层析柱安装在冷室中的溶胞产物 - 树脂混合物。允许未结合的蛋白质，通过重力柱。 </li><li>洗净，用300毫升的洗涤缓冲液I（的20mM Tris-盐酸，pH值7.4，400 mM氯化钠，1mM EDTA的溶液，pH值7.4和0.2mM的PMSF）的直链淀粉树脂。 </li><li>重复洗涤用150ml洗涤缓冲液II（20 mm T型的上升HCl，pH值7.4，200 mM氯化钠，1mM的EDTA，pH为7.4，为0.2mM PMSF）。 </li><li>加入5毫升的洗脱缓冲液（的20mM的Tris-HCl，pH值7.4，200 mM氯化钠，1mM EDTA的溶液，pH值7.4和0.2mM的PMSF，10mM的麦芽糖）柱中，温育在4℃下进行10分钟，收集洗脱液重复多次。 </li><li>合并洗脱液，确定通过加载后，以考马斯亮蓝染色（ 图4）上的SDS-PAGE凝胶的洗脱液的等分试样MBP-Mgm101的产率和纯度。 </li></ol> 3。断前蛋白酶裂解和阳离子交换层析<ol><li>添加50单位PreScission蛋白酶到的MBP-Mgm101洗脱液。在4℃下进行裂解位的CO / N，并允许它继续透析过程中对透析缓冲液（的的20mM Tris-HCl，pH值7.4，100 mM氯化钠，2mM的DTT，1mM EDTA的溶液，pH值7.4和0.2mM的PMSF） </li><li> SDS-PAGE凝胶上（ 图5A），检查裂解的效率。 </li><li>由多个注入负荷切割MBP Mgm101离子在生物秤迷你宏观准备高S墨盒连接到Bio-Rad公司生物DuoFlow FPLC系统。 </li><li>开始的阳离子交换色谱法，申请的步骤盐梯度为5mM，300mM的，500毫米，750毫米和1,000 mM NaCl的混合缓冲液A（5 mM氯化钠所建立的;​​的10mM磷酸钠，pH为7.2; mM的PMSF）和缓冲液B（1M NaCl的10mM的磷酸钠，pH为7.2，1mM的PMSF）。设置在0.5毫升/分钟的流速。收集Mgm101馏分和检查产品的纯度的蛋白在SDS-PAGE凝胶（ 图5B）。 </li></ol> 4。尺寸排阻色谱法<ol><li>结合从阳离子交换色谱的蛋白质部分。透析GF平衡缓冲液中的蛋白质（的20mM MOPS，pH为7.0，150 mM氯化钠，1mM的EDTA，pH值7.4，5mM的2 - 巯基乙醇，0.2mM的PMSF）。 </li><li> VIVASPIN 15R超滤离心柱浓缩蛋白，以3000×g在4℃下通过纺丝，以减低音量〜1毫升</li><li>装载Mgm101Ø呐校准SUPEROSE 6制备级柱层析缓冲液平衡过的20mM MOPS，pH为7.0，150 mM氯化钠，5mM的β-巯基乙醇，1mM EDTA的溶液，pH值7.4，0.2mM的PMSF。 </li><li>尺寸排阻色谱运行缓冲液，流速为0.5ml /分钟。 </li><li>收集的零碎，纯化Mgm101峰。负载的等分试样的级分在12％的SDS-PAGE检查蛋白质的最终质量。 </li><li>与VIVASPIN 6浓缩蛋白，然后迅速冻结样品在液氮中并储存在-80℃下</li><li>中使用Mgm101样品在6-12个月内单链DNA结合测定（ 图8），并用透射电子显微镜（ 图9）的结构的可视化。 </li></ol>

<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>

作者宣称，他们有没有竞争经济利益。

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 ...

preparation-mgm101-recombination-protein-mbp-based-tagging

Research

JoVE Journal

Biology

12.0K 次观看.  State University of New York Upstate Medical University. 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。

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

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

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

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

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

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

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

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

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

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

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 ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

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

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

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 ...

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

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

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 ...

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

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4&#039;,6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

秀丽线虫绿色荧光蛋白标记和 4 '、6-Diamidino-2-苯基吲哚染色检测蛋白质亚细胞定位

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

通过流动细胞测定检测和描述 Vivo 中的蛋白质自组装

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

一种用于快速鉴定根系分泌物中根细菌化学引诱剂的改进趋化性测定

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...