Name: using secm arrest sequence as a tool to isolate ribosome bound polypeptides
Uploaded: 2012-06-19T12:00:00
Description: Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

这项工作是由人类前沿科学计划授予RGP0024。

1。 DNA模板制备及体外转录<ol><li>感兴趣的基因被克隆在任何T7和/或如SP6启动基于质粒。为了获得利益的RNC，C-末端的多肽的目标是扩大加入逮捕诱导序列的SECM FXXXXWIXXXXGIRAGP 7。为了确保感兴趣的多肽片段，将挤出的核糖体隧道，目标蛋白C-末端的一部分被延长至少30个氨基酸12-14。可以推出一个灵活的富含甘氨酸，丝氨酸连接器之间的蛋白质和SECM逮捕序列，以避免任何可?...

<ol>
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对于重复性的结果，使用的组件， 在体外转录和翻译的质量和浓度是关键。我们已经用市售的体外转录和翻译提取的，他们给高效率和可重复性的结果，如果小心处理。质量可以影响mRNA的翻译，所以它是最重要的测试之前使用它在体外翻译的mRNA的完整性。 体外翻译的孵化时间随长度的蛋白质。此外，离心时间/速度可相应调整的情况下，70S核糖体的分数没有得?...

<table border="1"><tbody><tr><td> 试剂名称/盒 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> MEGAscript T7高产转录试剂盒</td><td> Ambion公司</td><td> AM1333 </td></tr><tr><td>转运站100大肠杆菌兴业套件</td><td> 5总理</td><td> 2401100 </td></tr><tr><td>核糖核酸酶抑制剂</td><td> Invitrogen公司</td><td> 15518012 </td></tr><tr><td>反式[35]标签</td><td> MP的Biomedicals </td><td> 0151006 </td></tr><tr><td> amicon超4离心过滤单元</td><td> Millipore公司</td><td> UFC801008 </td></tr><tr><td>存储荧光粉显影</td><td> GE医疗集团</td><td>台风9410可变模式成像</td></tr><tr><td>密度梯度分离系统</td><td> Teledyne公司ISCO​​公司</td><td>职业分类可编程密度梯度系统</td></tr><tr><td>蔗糖梯度岑trifugation </td><td>贝克曼</td><td>舰L - 90 K制备超速离心机</td></tr></tbody></table>

using secm arrest sequence as a tool to isolate ribosome bound polypeptides

广泛的研究提供了充足的证据表明，细胞中蛋白质折叠是合作转化过程1-5。不过，确切的途径来实现其功能的形式在合作转化折叠多肽链的如下仍然是一个谜。为了了解这个过程，以确定确切的翻译合作的折叠中间体构象，它是必不可少的开发技术，允许携带预定大小的新生链的RNC，让他们进一步的结构分析的隔离。 SECM（分泌显示器）是一个170个氨基酸E.大肠杆菌蛋白，调节下游SECA（SECM SECA操纵6分泌驾驶）在ATP酶的表达。 nakatogawa和伊藤最初发现了17个氨基酸组成的长序列中的SECM蛋白的C端区域（150-FSTPVWISQA QGIRAĞ的P-166）是足够的和必要的造成拖延在Gly165 SECM伸长，从而产生肽，甘氨酰-tRNA的稳定势必核糖体P位7-9。更重要的是，它被发现，这17个氨基酸的长序列可以融合到几乎所有的全长和/或截断，从而使生产的RNC携带新生链的预定大小7蛋白C-末端。因此，当融合或插入到目标蛋白质，SECM拖延序列产生的多肽链延伸逮捕，并在体内产生稳定在大肠杆菌中的RNC 大肠杆菌细胞和无细胞系统在体外 。进一步利用蔗糖密度梯度离心法分离的RNC。 孤立的RNC可以用来分析合作翻译的折叠中间体的结构和功能特点。最近，这项技术已成功地用于获得多个核糖体结合10,11新生链的结构见解。在这里，我们描述了隔离牛γ-B晶体RNCS融合SECM和在体外翻译系统生成的。

Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides (Video) | JoVE

我们在这里描述，现在经常使用的孤立稳定结合的核糖体新生肽链复合物（RNC）中的技术。这种技术发现了17个氨基酸长SECM“逮捕序列”可以停止在原核翻译延伸的优势（<em&gt; E。大肠杆菌</em&gt;）系统，几乎所有的蛋白质（或C-末端融合）插入时。

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

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