Name: assembly and purification of prototype foamy virus intasomes
Uploaded: 2018-03-19T15:00:00
Description: Watch this Scientific Journal Video about Assembly and Purification of Prototype Foamy Virus Intasomes at JoVE.com

这项工作得到了 NIH AI099854 和 AI126742 的支持。

1. vDNA 的退火
<ol><li>组合1X 十缓冲器 (10X 十库存: 100 毫米三盐酸, pH 值 8.0, 1 米氯化钠, 10 毫米 EDTA), 10 µM 寡聚 1 (5 ' ATTGTCATGGAATTTTGTATATTGAGTGGCGCCCGAACAG 3 ', 100 µM 股票) 和10µM 寡聚 2 (5 ' CTGTTCGGGCGCCACTCAATATACAAAATTCCATGACA 3 ', 100 µM 股票) 在最后容量1.5 毫升.整除25µL 六十0.2 毫升聚合酶链反应 (PCR) 管。 注: 根据应用的不同, 低聚糖可以用显影或标签在 5 '-末端的寡聚2或作为内部氨基 t 在基13从 5 '-端的寡聚1?...

<ol>
	<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>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>Hare, S., Gupta, S. S., Valkov, E., Engelman, A., Cherepanov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+intasome+assembly+and+inhibition+of+DNA+strand+transfer.">Retroviral intasome assembly and inhibition of DNA strand transfer.</a> Nature. 464 (7286), 232-236 (2010).</li><li>Li, M., Lin, S., 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=Outer+domains+of+integrase+within+retroviral+intasomes+are+dispensible+for+catalysis+of+DNA+integration.">Outer domains of integrase within retroviral intasomes are dispensible for catalysis of DNA integration.</a> Protein Sci. 25 (2), 472-478 (2016).</li><li>Gupta, 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=Solution+conformations+of+prototype+foamy+virus+integrase+and+its+stable+synaptic+complex+with+U5+viral+DNA.">Solution conformations of prototype foamy virus integrase and its stable synaptic complex with U5 viral DNA.</a> Structure. 20 (11), 1918-1928 (2012).</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>Lopez, M. A., Mackler, R. M., Altman, M. P., 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=Detection+and+removal+of+nuclease+contamination+during+purification+of+recombinant+prototype+foamy+virus+integrase.">Detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase.</a> J Vis Exp. , (2017).</li><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>Poirier, M. G., Bussiek, M., Langowski, J., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+access+to+DNA+target+sites+in+folded+chromatin+fibers.">Spontaneous access to DNA target sites in folded chromatin fibers.</a> J Mol Biol. 379 (4), 772-786 (2008).</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><li>Ballandras-Colas, A., 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=Cryo-EM+reveals+a+novel+octameric+integrase+structure+for+betaretroviral+intasome+function.">Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function.</a> Nature. 530 (7590), 358-361 (2016).</li><li>Ballandras-Colas, A., 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+supramolecular+assembly+mediates+lentiviral+DNA+integration.">A supramolecular assembly mediates lentiviral DNA integration.</a> Science. 355 (6320), 93-95 (2017).</li><li>Passos, D. O., 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=Cryo-EM+structures+and+atomic+model+of+the+HIV-1+strand+transfer+complex+intasome.">Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.</a> Science. 355 (6320), 89-92 (2017).</li><li>Yin, Z., 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+the+Rous+sarcoma+virus+intasome.">Crystal structure of the Rous sarcoma virus intasome.</a> Nature. 530 (7590), 362-366 (2016).</li><li>Maertens, G. N., Hare, S., Cherepanov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+retroviral+integration+from+X-ray+structures+of+its+key+intermediates.">The mechanism of retroviral integration from X-ray structures of its key intermediates.</a> Nature. 468 (7321), 326-329 (2010).</li><li>Maskell, D. 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=Structural+basis+for+retroviral+integration+into+nucleosomes.">Structural basis for retroviral integration into nucleosomes.</a> Nature. 523 (7560), 366-369 (2015).</li></ol>

逆转录病毒的 multimeric 与病毒基因组 DNA 进行整合, 并继续病毒生命周期。每 intasome 单体的数量可以是 tetramers、octamers 或可能更高的顺序多聚体11, 12, 13, 14.PFV intasomes 是聚丙烯重组的双链 DNA 寡聚物, 模仿病毒基因组 dna 结束3.这些 intasomes 已用于结构和动态研究3</s...

将病毒基因组集成到宿主基因组是所有逆转录1生命周期中的一个强制性步骤。病毒酶整合酶 (in) 催化病毒 DNA 基因组的每一端的共价键连接到宿主 DNA。在细胞感染期间, 它是一个整合整合的预集成复合体的一部分。重组复合双链 DNA 寡聚物模拟病毒 DNA 末端也可以进行集成到目标 DNA体外2。一个共同的综合试验体外利用超螺旋质粒作为靶 DNA。将两种病毒 DNA 寡聚物 (vDNA) 与质粒整合在一起会产生线性乘积, 并被称为协同集成 (图 2A)。集成化验体外也可能产生的产品只有一个 vDNA 共价键加入到目标质粒, 导致一个松弛的循环。这种半站点集成产品似乎是检测体外的一个伪品。
重组和 vDNA 可能执行集成在体外, 但它们不是理想的试剂, 研究的动力学或结构的集成络合物时, 单体将模糊相关的可视化。纯集成复合物, 或 "intasomes", 是动态单分子分析或结构研究所必需的。PFV 和 vDNAs 可以通过透析从一个相对较高的盐浓度缓冲, 以较低的盐浓度3,4。在透析过程中, 形成沉淀形式。这种沉淀从透析中除去, 盐浓度增加。盐浓度越高, 含 PFV intasomes 的沉淀 solubilizes。然后用尺寸排除色谱 (SEC) 纯化 intasomes。重组原型泡沫病毒 (PFV) 已被证明存在作为单体在溶液中的浓度高达225µM5。SEC 分离有效地分离 PFV intasomes (225.5 kDa), 其中包括 PFV 的聚丙烯和二 vDNAs, 从单体 PFV 在 (44.4 kDa) 和自由 vDNA (24.0 kDa)。PFV intasomes 可能会被冻结, 并在-80 ˚C 至少保存六月的集成活动。
重组 PFV intasomes 也可能被修改, 包括在氨基酸替代或截断突变或 vDNAs 标记为显影或生物素4,6。纯化后的 PFV intasomes 能很容易地整合成超螺旋质粒靶 DNA体外。intasomes 的散装生化积分化验可以测试突变, 抑制剂, 或其他化学添加剂的影响。生物素化 intasomes 可以用来探测与核酸或蛋白质的亲和性。PFV intasomes 在室温下功能, 允许用磁镊进行单分子显微分析, 测量两个 vDNA 端或全内反射荧光之间的连接时间, 以可视化目标的 intasome 搜索。DNA6。此外, PFV intasomes 是第一个结构上具有显著影响的逆转录病毒集成领域3。

<table>
	<tbody>
		<tr>
			<td>DNA Oligomers</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Custom DNA Oligos</td>
		</tr>
		<tr>
			<td>Tris Ultra Pure</td>
			<td>Gojira Fine Chemicals</td>
			<td>UTS1003</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>P212121</td>
			<td>RP-S23020</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure EDTA</td>
			<td>Invitrogen/Gibco</td>
			<td>15575</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 mL centrifugal filters</td>
			<td>Sigma-Aldrich</td>
			<td>Z677094-24EA</td>
			<td>3 kDa MWCO</td>
		</tr>
		<tr>
			<td>DTT&#160;</td>
			<td>P212121</td>
			<td>SV-DTT</td>
			<td></td>
		</tr>
		<tr>
			<td>BIS-TRIS propane,&#62;=99.0% (titration)</td>
			<td>Sigma-Aldrich</td>
			<td>B6755-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>208086</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Amresco</td>
			<td>0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>G37-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel-loading tips, 1 - 200 &#956;L</td>
			<td>Corning</td>
			<td>CLS4853-400EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade; Single-edged; 100/Pk.; Pack of 100</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Filter Units with PES Membrane &#62; 250mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>568-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing clips</td>
			<td>Spectrum Labs</td>
			<td>132734</td>
			<td></td>
		</tr>
		<tr>
			<td>6-8 kDa 10 mm Dialysis Tubing</td>
			<td>Spectrum Medical</td>
			<td>132645</td>
			<td></td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17517201</td>
			<td></td>
		</tr>
		<tr>
			<td>Hi-Res Standard Agarose</td>
			<td>AGTC Bioproducts</td>
			<td>AG500D1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1302</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Fisher Scientific</td>
			<td>0-267</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyladder 10kb, 500 lanes</td>
			<td>Denville Scientific</td>
			<td>CB4225-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

assembly and purification of prototype foamy virus intasomes

反转录病毒生命周期的一个定义特征和必要步骤是将基因组整合到宿主基因组中。所有逆转录编码一个整合酶 (in) 酶, 催化共价连接病毒的宿主 DNA, 这被称为链转移。集成可以建模体外与重组逆转录病毒和 DNA 寡聚物模仿病毒基因组的两端。为了更仔细地重述发生体内的集成反应, 在减少的盐浓度缓冲液中, 通过透析在重组和合成寡聚物中组装出集成络合物。集成复合体称为 intasome, 可通过尺寸排除色谱法纯化。在原型泡沫病毒 (PFV) 的情况下, intasome 是一个聚丙烯和两个 DNA 寡聚物, 是容易分离的单体和游离寡聚 dna。PFV intasomes 的整合效率可以在各种实验条件下进行测定, 以更好地了解逆转录病毒集成的动力学和机理。

PFV intasomes 由重组和 vDNA 组装而成。程序集后, intasomes 由 SEC (图 1) 进行纯化。用超螺旋 DNA 靶和琼脂糖凝胶电泳 (图2) 测定各分数的积分活性。这种凝胶是成像与荧光扫描仪设置, 以检测 EtBr (和荧光, 如果使用荧光标记 vDNA)。使用图像分析软件, 可以使用带像素卷来计算集成效率 (图 3)。积分效率最高的分数...

Watch this Scientific Journal Video about Assembly and Purification of Prototype Foamy Virus Intasomes at JoVE.com

Assembly and Purification of Prototype Foamy Virus Intasomes

重组逆转录病毒整合酶和 dna 寡聚物模仿病毒 dna 末端可以形成一个酶活跃的复杂称为 intasome。Intasomes 可用于生物化学、结构和动力学研究。本协议详细介绍了如何组装和纯化原型泡沫病毒 intasomes。

泡沫病毒 Intasomes 原型的组装与纯化

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