Name: detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase
Uploaded: 2017-12-08T14:00:00
Description: Watch this Scientific Journal Video about Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase at JoVE.com

这项工作得到了 NIH AI099854 和 AI126742 的支持。

1. 诱发 PFV 在大肠杆菌中的表达式
<ol><li>
添加1μ l 的 PFV 在表达质粒 (10 ng/μ l) 到20μ l 的大肠杆菌BL21 (DE3) pLysS (20 μ l 分的商业可用细胞有 > 2 x 106 CFU/微克质粒) 在1.5 毫升管。用手指轻轻敲击或轻拂管子。在冰上孵育5分钟。
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<li>热休克整整三十年代在42˚C 水浴, 然后立即返回到冰2分钟。</li>
		<li>添加80μ l 室温超优化汤与物抑制 (SOC) 媒体。孵育?...

<ol>
	<li>Lopez, M. A., Mackler, R. M., Yoder, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+nuclease+contamination+during+purification+of+recombinant+prototype+foamy+virus+integrase.">Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase.</a> J Virol Methods. 235, 134-138 (2016).</li><li>Senavirathne, G., 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=Widespread+nuclease+contamination+in+commonly+used+oxygen-scavenging+systems.">Widespread nuclease contamination in commonly used oxygen-scavenging systems.</a> Nat Methods. 12 (10), 901-902 (2015).</li><li>Gunn, K. H., Marko, J. F., Mondragon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthogonal+single-molecule+experiment+reveals+multiple-attempt+dynamics+of+type+IA+topoisomerases.">An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases.</a> Nat Struct Mol Biol. 24 (5), 484-490 (2017).</li><li>Valkov, E., 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+characterization+of+the+integrase+from+the+prototype+foamy+virus.">Functional and structural characterization of the integrase from the prototype foamy virus.</a> Nucleic Acids Res. 37 (1), 243-255 (2009).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Li, M., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+viral+DNA+ends+channels+the+HIV-1+integration+reaction+to+concerted+integration.">Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration.</a> J Biol Chem. 280 (32), 29334-29339 (2005).</li><li>Li, M., Mizuuchi, M., Burke, T. R., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+DNA+integration:+reaction+pathway+and+critical+intermediates.">Retroviral DNA integration: reaction pathway and critical intermediates.</a> EMBO J. 25 (6), 1295-1304 (2006).</li><li>Sinha, S., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+immunodeficiency+virus+type+1+integrase+exhibits+a+capacity+for+full-site+integration+in+vitro+that+is+comparable+to+that+of+purified+preintegration+complexes+from+virus-infected+cells.">Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells.</a> J Virol. 79 (13), 8208-8216 (2005).</li><li>Goodarzi, G., Im, G. J., Brackmann, K., Grandgenett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+integration+of+retrovirus-like+DNA+by+human+immunodeficiency+virus+type+1+integrase.">Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.</a> J Virol. 69 (10), 6090-6097 (1995).</li><li>Vora, A. C., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+catalytic+properties+of+retrovirus+integrase-DNA+complexes+capable+of+efficiently+performing+concerted+integration.">Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.</a> J Virol. 69 (12), 7483-7488 (1995).</li><li>Jones, N. D., 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=Retroviral+intasomes+search+for+a+target+DNA+by+1D+diffusion+which+rarely+results+in+integration.">Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.</a> Nat Commun. 7, 11409 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li></ol>

与 dna 相互作用的重组蛋白, 如 dna 修复蛋白, 单分子显微镜应用的氧清除剂, 或逆转录病毒 integrases, 应无污染细菌核酸2,3。这些污染物可能会混淆的解释结果在散装生物化学或单一分子化验。
我们发现, 细菌核酸经常与 PFV 一起净化。然而, PFV 在显示一种亲和力的肝素琼脂, 是容易区分的细菌核酸污染物1。制备无核...

蛋白质与 DNA 相互作用的生物化学和单分子研究需要格外纯的重组蛋白。污染核酸的细菌可以掩盖这些化验结果。从大肠杆菌(大肠杆菌) 中分离出的重组蛋白氧清除 protocatechuate-3,4-dioxygenase (PCD) 和原型泡沫病毒 (PFV) 整合 (in) 的制备中发现了污染核酸1,2,3。
逆转录病毒集成检测依赖于超 DNA 到有缺口或线性产品的转换, 作为活动的4。在细胞感染期间, 将病毒 cDNA 的两端连接到宿主染色质5。每个端接反应称为链转移。重组活性的测定可以加入两个 dna 齐聚物, 模仿病毒 cDNA 结束到目标 dna 在一致的集成反应中4,5,6,7,8。二者择一地重组在5月加入仅一个脱氧核糖核酸末端在一个非生理相关的半站点综合反应9,10。当超质粒 dna 是整合的目标时, 协同集成产品是线性化 dna 和半站点集成产品的放松圈。这些反应产物通过其相对流动性在琼脂糖凝胶电泳1。如果重组中有一个污染的核酸, 将有假放松圈或可能线性化的质粒混淆了实验结果。病毒 DNA 寡聚物可能被荧光标记, 以最终确定集成产品, 而不是核酸产品。然而, 在很大程度上有利于超的 DNA 靶点;任何超质粒通过污染核酸而导致的松弛圆或线性 DNA 的损失都可能扭曲数据11的结果和解释。因此, 在制剂中清除逆转录病毒的细菌核酸是势在必行的。
PFV 对肝素琼脂有不同的亲和力, 与细菌核酸1比较。PFV 和核酸可由肝素琼脂的线性梯度洗脱分离。核酸在 280 nm 峰值或通过分析十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 的紫外线 (UV) 吸收后不易检测到。相反, 核酸是通过核酸活性测定检测的, 采用超质粒转化为松弛的圆或线性产品。肝素琼脂色谱法测定核酸活性。PFV 和核酸污染物对单 Q 阴离子树脂亲和性无影响。单 S 阳离子树脂亲和性有很小的差异。然而, 细菌核酸和 PFV 的单 S 分辨率不会允许有效分离蛋白质。最终, 肝素琼脂亲和纯化提供了最好的分离细菌核酸从 PFV 在和具有无限的优势的负荷量。
对污染核酸活性的检测可以适应其他蛋白质。兴趣的蛋白质可能有其他的亲和力特征比 PFV 在;有兴趣的蛋白质和核酸污染物的结合特征的区别必须是经验主义地确定的。这种识别核酸污染的方法可适用于其他树脂, 包括单 S 阳离子或单 Q 阴离子交换树脂。亲和性和离子交换树脂可以提供一种可靠的方法来分离核酸污染的重组蛋白, 而不限制色谱中蛋白质的体积。

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detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase

逆转录病毒原型 (PFV) 中的整合蛋白是研究反转录酶的机制。与其他病毒相比, PFV 在更易于溶解, 更适于实验操作。此外, 它是敏感的临床相关的人体免疫缺陷病毒 (HIV-1) 的抑制剂, 表明 PFV 的催化机制类似于 HIV-1 在. 在催化病毒互补 DNA (cDNA) 共价加入靶DNA 在一个称为链转移的过程中。这股转移反应引入了缺口的目标 DNA。综合反应产品的分析可以被混淆由核酸的存在相似地尼克脱氧核糖核酸。在大肠杆菌(大肠杆菌) 中, 一种细菌核酸已被证明与重组 PFV 联合纯化.在这里, 我们描述了一种方法, 以隔离 PFV 从污染核酸的肝素亲和层析。通过超质粒和琼脂糖凝胶电泳, 可以很容易地筛选出分数核酸污染。PFV 在和污染的核酸显示替代亲和力的肝素琼脂允许核酸自由制备重组 PFV 适合散装生物化学或单分子分析一体化。

重组 PFV 中经常被细菌核酸1污染。生化积分测定取决于超质粒 DNA 转化为松弛圆和线性产品的定量。污染核酸的存在可能导致这些化验的虚假定量。在大肠杆菌(图 1) 中, 首先用镍亲和色谱 (图 2) 对 PFV 的表达式进行了 hexahistidine 标记。与几乎纯 PFV 的分数相结合, hexahistidine 标记由蛋白酶劈开。我们已经确定, PFV 和污染核酸有不?...

Watch this Scientific Journal Video about Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase at JoVE.com

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

重组原型泡沫病毒整合蛋白经常被细菌核酸在纯化过程中污染。这种方法识别核酸污染, 并将其从酶的最终制备中除去。

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

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