Name: detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase
Uploaded: 2017-12-08T14:00:00.000+00:00
Duration: 7 min 50 s
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。
	<ol>
<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。孵育为1小时在37˚C 以225转每分钟 (rpm) 震动。</li>
		<li>25μ l 的混合物在100毫米 x 15 毫米培养皿中, 含有25毫升的 Luria 肉汤 (lb) 琼脂 (1 L 磅琼脂:10 克 bacto-胰, 10 克氯化钠, 5 克酵母提取物, 15 克琼脂) 和100微克/毫升氨苄西林 (100 毫克/毫升的库存溶液)。</li>
		<li>在第二个相同的板上将剩余的75μ l 转化后的细胞板。在37° c 的夜间, 用盖子将盘子孵育。</li>
	</ol>
</li>
	<li>
检索已转换的大肠杆菌的板.使用一个单一的殖民地接种3毫升磅 (1 升磅:10 克 bacto-胰, 10 克氯化钠, 5 克酵母提取物) 补充100微克/毫升氨苄西林在14毫升圆底聚丙烯培养管。孵育为〜8小时在37˚C 与 225 rpm 晃动。
	<ol>
<li>在34毫升锥形瓶中加入100微克/毫升氨苄西林和34微克/毫升氯霉素 (250 毫克/毫升的库存溶液), 添加3毫升的养殖50毫升磅。在37° c 的夜间孵育培养, 225 rpm 晃动。</li>
		<li>将53毫升的养殖转移到1升磅、100微克/毫升氨苄青霉素和34微克/毫升氯霉素在4升锥形瓶中。孵育在37° c 与225转每分钟摇晃。</li>
		<li>定期测量区域性的 600 nm (OD600) 的光学密度。最初, 检查每个 h 的区域性 OD600 。当区域性达到所需的 OD600时, 请检查每15分钟不满。将区域性扩展到0.9 和1.0 之间的 OD600 。将烧瓶转到冰浴和旋流中, 以降低养殖温度。</li>
	</ol>
</li>
	<li>
将1毫升的培养基转移到1.5 毫升的试管中, 然后进行变性 SDS-page 分析。
	<ol>
<li>将1.5 毫升管中的细胞在室温下离心1分钟, 在离心 1.4万 x g。丢弃上清液。重150μ l 磷酸缓冲盐 (PBS) 中的颗粒移。PBS 可以是有或没有 CaCl2和氯化镁2。</li>
		<li>添加150μ l 2x sds-页样品缓冲液 (150 毫米三盐酸, pH 6.8, 1.2% sds, 30% 甘油, 15% β-基 (βME), 0.0018% 溴蓝色), 并通过吸管或涡流彻底混合。在-20 ° c 下煮沸3分钟。</li>
	</ol>
</li>
	<li>添加到细菌培养: 最后浓度0.25 毫米异丙基β-d-thiogalactopyranoside (IPTG, 0.25 米的股票溶液) 和50μ m ZnCl2 (50 mM 股票解决方案)。IPTG 诱导表达的 PFV在基因从 T7 启动子和 ZnCl2是补充为正确折叠的一个氨基终端锌指域在 PFV. 孵育文化在25° c 以震动在 225 rpm 为 4 h. 立刻把烧瓶转到冰浴。保存1毫升的文化为 SDS-页分析遵循相同的议定书, 如以上1.3。</li>
	<li>
将细菌培养转化为适当大小的离心瓶。例如, 1 L 文化可以转移到四无菌一次性250毫升锥形底聚丙烯瓶。旋转的瓶子在一个冷藏桌面离心机在 3000 x g 20 分钟在4˚C。通过浇注丢弃清。
	<ol>
<li>重所有的颗粒从 1 L 细菌培养在总容量25毫升 (6.25 毫升每瓶) 冷 PBS (有或没有 CaCl2和氯化镁2) 由移。将细菌悬浮液与一个50毫升的锥形管结合并转移。旋转在一个冷冻离心机在 3000 x g 为20分钟在4° c。通过浇注或移丢弃上清液。</li>
	</ol>
</li>
	<li>重在10毫升悬浮缓冲 (50 毫米三盐酸, pH 7.5, 500 毫米氯化钠, 和500μ m phenylmethylsulfanoxide (PMSF) 蛋白酶抑制剂) 移的细菌颗粒。通过将试管放入带有液态氮气的杜瓦瓶中, 使其凝固, 足以淹没50毫升管。小心地将管子从液氮中取出, 贮存在-80 ° c。 注: 细菌颗粒可在-80 ° c 贮存数月。在-80 ° c 贮藏两年后, 我们没有发现活性蛋白的损失。</li>
	<li>
用8.3 厘米宽, 7.3 厘米高, 0.75 毫米厚的 SDS-页状凝胶, 1.5 毫升6% 聚丙烯酰胺堆叠层 (125 mm 三盐酸, pH 6.8, 6% 丙烯酰胺, 0.1% SDS, 过硫酸铵, 0.1% TEMED) 和 0.001% ml 3.5 分析预感应和后诱导样品聚丙烯酰胺解决层 (375 毫米三盐酸, pH 8.8, 10% 丙烯酰胺, 0.1% SDS, 过硫酸铵, 0.1% TEMED)0.001%。
	<ol>
<li>倒入堆叠凝胶后, 插入10井梳。当凝胶有聚合, 组装页设备, 并添加足够的 SDS 页运行缓冲 (25 毫米三, 192 毫米甘氨酸, 0.1% sds)。</li>
		<li>装载10μ l 每个样品和4μ l prestained 蛋白质标准。运行在16.5 伏/厘米, 直到染料前到达底部的凝胶, 约60分钟。</li>
		<li>拆开凝胶板, 将凝胶转移到塑料浴缸。添加马斯亮明亮的蓝色染色液 (0.1% 马斯亮亮蓝色 R-250, 40% 甲醇, 10% 乙酸) 慷慨地覆盖在室温下20分钟的凝胶和染色。</li>
		<li>通过浇注和更换 destain 溶液 (40% 甲醇, 10% 醋酸) 除去染色液, 直到带很容易看到。通过浇注和更换去离子水去除脱色溶液。PFV 在后诱导样品中应易于识别。PFV 与 hexahistidine 标签有一个分子量 47374 Da。</li>
		<li>如果 PFV 不可见, 则从一个变换板中的一个不同的菌落开始重复感应。</li>
	</ol>
</li>
</ol>2. 镍亲和层析
<ol><li>在冰上解冻一个细菌颗粒。 注意: 这将需要很大的时间 (约 2-3 小时)。当颗粒解冻到一个细胞浆, 添加一个额外的500μ m PMSF (100 毫米库存溶液)。</li>
	<li>将50毫升的细菌浆管放在冰上。几种的细菌在30% 振幅为三十年代 (1 s, 1 s 关闭) 每丸从 1 L 培养使用 sonicator 与0.5 英寸直径的生物喇叭与0.125 英寸直径锥形 mircotip, 频率20赫和最大功率 400 W。</li>
	<li>将细胞样品转移到冷 ultracentrifuge 管。使用聚碳酸酯瓶组件 (25 毫米直径, 89 毫米高度) 与 a 型60钛固定角转子兼容。如果达到相同的引力力, 可选用其它管和转子。旋转 12万 x g 为60分钟在4° c。应该有一个明显的颗粒。上清的颜色可能是黄色的。</li>
	<li>将上清液倒入或移到冷的50毫升锥形管上。注意音量;此卷应近似于在对齐冻结之前使用的悬浮缓冲区的体积。如果上清液粘稠, 可能会被细菌 DNA 污染, 从而堵塞色谱柱。在这种情况下, 重复离心, 以颗粒的细菌 DNA。</li>
	<li>
准备500毫升缓冲器 A (50 毫米三盐酸, ph 7.5, 500 毫米氯化钠, 500 μ m PMSF) 和500毫升缓冲 B (50 毫米三盐酸, ph 7.5, 500 毫米氯化钠, 500 μ m PMSF, 200 毫米咪唑)。 注意: 在本协议中, 将快速蛋白质液相色谱 (FPLC) 系统、所有缓冲器和样品保存在4° c 的冷室中。<ol>
<li>无菌过滤器缓冲器 A 和 B 与0.2 μ m 一次性过滤器单位。根据 FPLC 系统, 分别用缓冲器 a 和缓冲器 b 清洗泵 a 和泵 b。流90% 缓冲器 a 和10% 缓冲器 B 通过系统, 直到电导率和 UV 读数稳定。最大流速将取决于仪器;使用5毫升/分钟的流量, 最大压力限制为1.0 兆帕斯卡。</li>
	</ol>
</li>
	<li>准备一个110毫米长, 5 毫米直径 FPLC 柱与1.5 毫升镍充电树脂 (最大结合容量50毫克/毫升, 最大压力 1 MPa)。该列可在提纯前的前一天准备好, 贮存在4˚C。</li>
	<li>将列连接到 FPLC 仪器。平衡20毫升的90% 缓冲器 a 和10% 缓冲 B (20 mM 咪唑) 的流量为0.5 毫升/分钟, 最大压力极限为0.5 兆帕斯卡。该仪器应收集 280 nm (一个280) 的电导率和紫外线吸收的实时数据。电导率和 UV 读数应稳定。如果这些读数没有稳定, 继续通过该列的流动缓冲区, 直到它们是稳定的。</li>
	<li>
在0.15 毫升/分钟的流速下, 将蛋白质样本 (每粒10毫升) 加载到柱上, 最大压力极限为0.5 兆帕斯卡。在整个净化过程中, 流动速率和柱压力保持不变。收集在50毫升管的流量。
	<ol>
<li>用20毫升90% 缓冲器 a 和10% 缓冲液冲洗柱 b. 在50毫升的管子里收集洗涤。洗蛋白质与 20 mL 线性梯度10% 到100% 缓冲 B (20 毫米到200毫米咪唑)。</li>
		<li>最后, 用5毫升100% 缓冲 B (200 mM 咪唑) 清洗柱。在0.27 毫升分数中收集渐变和最终洗涤。
</li>
	</ol>
</li>
	<li>
结合15μ l 2x SDS-页样本缓冲区与15μ l 的流动, 洗涤, 并峰值一个280分数。将样品煮沸3分钟。
	<ol>
<li>准备两 10% SDS 页凝胶, 如前所述的步骤 1.6, 除了15井梳子。将每个样品的10μ l 载入凝胶和4μ l prestained 蛋白标准。如步骤1.6 所述, 运行、着色和分析凝胶。</li>
		<li>在视觉检查的基础上, 将似乎包含几乎纯 PFV 的分数组合在一起。通过移测量体积, 通常为 8-10 毫升。</li>
		<li>使用分光光度计测量组合分数的一个280 , 并计算蛋白质中 PFV 的总量;我们的典型产量是每升10毫克的诱导培养。PFV 的消光系数与 hexahistidine 标记为 59360 cm-1 M-1。</li>
	</ol>
</li>
	<li>要删除 hexahistidine 标签, 补充 PFV 在10毫米糖 (德勤, 1 米的股票解决方案) 和0.1 毫米 EDTA, pH 8.0 (0.5 米库存解决方案)。添加15微克的人鼻 (HRV) 3C 蛋白酶每毫克 PFV 在. 孵育在4˚C 过夜。从 47374 da 到 44394 da, 裂解减少了分子量的 PFV, 并可通过 10% SDS 页分析进行验证。</li>
</ol>3. 肝素亲和层析
<ol><li>
准备250毫升缓冲剂 C (50 毫米三盐酸, ph 7.5, 10 毫米的, 0.1 毫米 edta, 500 μ m PMSF) 和250毫升缓冲 D (50 毫米三 hcl, ph 7.5, 10 毫米的数字式, 0.1 毫米 edta, 500 μ m PMSF, 1 米氯化钠)。 注: 在本协议中, FPLC 系统、缓冲器和样品保存在4˚C 的冷室中。<ol>
<li>无菌过滤器缓冲 C 和 D 与0.2 μ m 一次性过滤器单位。根据 FPLC 系统, 分别清洗泵 a 和泵 B 与缓冲器 C 和缓冲器 D。流80% 缓冲器 C 和20% 缓冲器 D 通过系统, 直到电导率和 UV 读数稳定。最大流速将取决于仪器;我们经常使用的流量为5毫升/分钟, 最大压力限制为1.0 兆帕斯卡。</li>
	</ol>
</li>
	<li>
准备一个80毫米长, 5 毫米直径 FPLC 柱与1毫升肝素琼脂树脂 (最大结合能力2.0 毫克的牛抗凝血酶 III 每毫升树脂; 最大压力 1.4 MPa)。该柱可在提纯前的前一天准备好, 贮存在4° c。将列连接到 FPLC 仪器。
	<ol>
<li>平衡20毫升的80% 缓冲 C 和20% 缓冲 D (200 毫米氯化钠) 的流量为0.5 毫升/分钟, 最大压力限制为1兆帕斯卡。该仪器应收集 280 nm (一个280) 的电导率和紫外线吸收的实时数据。电导率和 UV 读数应稳定。如果这些读数没有稳定, 继续通过该列的流动缓冲区, 直到它们是稳定的。</li>
	</ol>
</li>
	<li>通过增加1.5 卷缓冲 C, 将 PFV 的氯化钠浓度降低到200毫米。例如, 在中添加15毫升的缓冲 C 到10毫升 PFV。我们的总容量是典型地 ~ 25 毫升。</li>
	<li>
将稀释后的 PFV 在样品中, 以0.5 毫升/分钟的流速, 以最大压力极限1.0 兆帕斯卡的速度载入肝素琼脂柱。在整个净化过程中, 流动速率和柱压力保持不变。收集在50毫升锥形管的流量。
	<ol>
<li>用20毫升80% 缓冲 C 和20% 缓冲 D (200 毫米氯化钠) 冲洗柱, 在50毫升锥形管中收集洗涤液。洗 30 mL 线性梯度20% 到100% 缓冲 D (200 毫米到 1 M 氯化钠) 的专栏。</li>
		<li>用5毫升100% 缓冲器清洗柱 d. 在0.37 毫升分数中收集梯度和最终洗涤。</li>
	</ol>
</li>
	<li>分析负载, 流经, 洗涤和分数 8% SDS 页, 如所述, 在2.9。PFV 在以下的 hexahistidine 标签的裂解有一个分子量的 44394 Da 和灭绝系数保持59360厘米-1 M-1没有 hexahistidine 标记。将所有分数存储在4˚C, 直到完成核酸检测。</li>
</ol>4. 核酸测定
<ol><li>
确定肝素琼脂分数, 似乎含有几乎纯 PFV. 结合3μ l 肝素分数和 50 ng 3 kb 超质粒 DNA 在反应缓冲 (10 毫米 HEPES, pH 7.5, 110 毫米氯化钠, 5 mm MgSO4, 4 μ m ZnCl2, 10 毫米的数码化), 最后的体积为15μ l。孵育在37˚C 为90分钟, 包括一个没有 PFV 的负控制。
	<ol>
<li>停止反应与1毫克/毫升蛋白酶 K (20 毫克/毫升的股票溶液) 和 0.5% SDS (10% 库存溶液)。孵育在37° c 为 1 h. 样品可贮存在-20 ° c 供以后分析。</li>
	</ol>
</li>
	<li>
准备一个125毫升凝胶1% 重量每体积 (瓦特/v) 琼脂糖在1x 泰式缓冲器 (40 毫米三醋酸盐, 1 毫米 EDTA) 与0.1 µg/毫升溴溴化物 (10 毫克/毫升库存解决方案)13。融化琼脂糖溶液, 倒入15厘米 x 10 cm 凝胶铸造托盘。插入一个15井梳子与5毫米宽, 0.75 毫米厚实的井。与1.5 毫米厚井相比, 薄井具有更好的带分辨率。允许凝胶在室温下凝固。
	<ol>
<li>用0.1 µg/毫升溴溴化液浸泡在1x 泰的凝胶中。加入3.5 μ l 6x 负载染料 (50% 甘油, 0.15% 橙 G 染料) 的核酸测定反应。将整个卷装入凝胶。在恒定电压下运行凝胶, 10 伏每 cm (100 V) 在环境温度为 1 h 或直到橙色 G 染料前面到达胶凝体的末端。</li>
	</ol>
</li>
	<li>
立即图像琼脂糖凝胶与荧光扫描仪设置检测溴溴化物。线性 dna 应该以 3 kb 的大小运行, 超的 dna 应该跑得更快 (〜 2 kb), 而松弛的圆 dna 应该运行得更慢 (〜 3.5 kb)。
	<ol>
<li>利用图像分析软件, 计算各车道超、线性、松弛圆质粒的像素体积。调用像素卷的总和为这三脱氧核糖核酸形成 "总脱氧核糖核酸" 价值并且使用它计算线性和宽松圈子的百分比。例如, 松弛圆的像素体积除以该车道中的总 DNA 像素值, 乘以这个数100来确定一个百分比。含有污染核酸的分数与负控制相比, 显示的线性和松弛圆的百分比更高。</li>
	</ol>
</li>
	<li>结合肝素琼脂分数, 不显示污染核酸活动。透析在10毫米 6-8 kDa 分子量截止 (分子) 透析管在4° c 过夜对1毫米的 50 L 的-HCl, pH 7.5, 200 毫米氯化钠, 50 μ m ZnCl 2, 10 毫米.</li>
	<li>从透析管中回收样品中的无核酸 PFV。测量一个280并计算最终的蛋白质浓度。液氮中的分和冻结。商店等分在-80 ° c。</li>
</ol>

<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>

作者没有什么可透露的。

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。

<table><tbody><tr><td>BL21/DE3 Rosetta E. coli</td><td>EMD Millipore</td><td>70956-4</td><td></td></tr><tr><td>LB broth</td><td>EMD biosciences</td><td>1.10285.0500</td><td></td></tr><tr><td>Ampicillin</td><td>Amresco</td><td>0339</td><td></td></tr><tr><td>Chloramphenicol&amp;nbsp;</td><td>Amresco</td><td>0230</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>IPTG&amp;nbsp;</td><td>Denville Scientific</td><td>CI8280</td><td></td></tr><tr><td>ZnCl2</td><td>Sigma-Aldrich</td><td>208086</td><td></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>PMSF&amp;nbsp;</td><td>Amresco</td><td>0754</td><td></td></tr><tr><td>Imidazole&amp;nbsp;</td><td>Sigma-Aldrich</td><td>I0250</td><td></td></tr><tr><td>DTT&amp;nbsp;</td><td>P212121</td><td>SV-DTT</td><td></td></tr><tr><td>UltraPure EDTA</td><td>Invitrogen/Gibco</td><td>15575</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Amresco</td><td>0662</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Denville Scientific</td><td>CA3510</td><td></td></tr><tr><td>Ethidium bromide</td><td>Thermo Fisher Scientific</td><td>BP1302</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G37-20</td><td></td></tr><tr><td>Ni-NTA Superflow</td><td>Qiagen</td><td>30430</td><td></td></tr><tr><td>Heparin Sepharose 6 Fast Flow</td><td>GE Healthcare Life Sciences</td><td>17-0998-01</td><td></td></tr><tr><td>HRV14 3C protease&amp;nbsp;</td><td>EMD Chemicals</td><td>71493-3</td><td></td></tr></tbody></table>

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 ...

detection-removal-nuclease-contamination-during-purification

Research

JoVE Journal

Immunology and Infection

7.0K 次观看.  The Ohio State University College of Medicine. 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。

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

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

科研

免疫与感染

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

生物学

InSET: A Novel Tool for Unbiased Genome-Wide Functional Element Discovery

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

Insertional mutagenesis is an essential tool in forward genetics for identifying functional genomic elements. Here, we describe the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for detecting lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library.

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens ...

遗传学

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

通过单步肝素亲和层析纯化 AAV 载体

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

作者聚焦：腺相关病毒载体生产和纯化的改进方法

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

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

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流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

A型流感病毒的核蛋白复合物的分离纯化及可视化

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

泡沫病毒 Intasomes 原型的组装与纯化

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体