Name: in vitro reconstitution of the active t. castaneum telomerase
Uploaded: 2011-07-14T13:00:00
Description: Watch this Scientific Journal Video about In vitro Reconstitution of the Active T. castaneum Telomerase at JoVE.com

这里介绍的研究是由宾夕法尼亚州的卫生署，埃利森医学，V和祖母绿基础支持。

1。表达的重组TC叔<ol><li>从六个新鲜板用的TEV裂解N -端含有合成TC叔质粒转化罗塞塔（DE3）pLysS中细胞接种6升（6 × 2升困扰锥形瓶包含在每1 ​​L 2YT肉汤）2YT媒体hexahistidine标签。 </li><li>细胞增长在37 ° C，而在220转的0.5-0.6一个外径600的摇晃。 </li><li>加入1 mM的IPTG诱导蛋白表达。 </li><li>降低温度至30 ° C，缓慢摇动至150转，让细胞表达蛋白质为4-5小时</li><li>离心收集细胞在4 ?...

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	<li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT.">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455, 633-637 (2008).</li><li>Greider, C. W., Blackburn, 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=Identification+of+a+specific+telomere+terminal+transferase+activity+in+Tetrahymena+extracts.">Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.</a> Cell. 43, 405-413 (1985).</li><li>Harley, C. B., Futcher, A. B., Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+shorten+during+ageing+of+human+fibroblasts.">Telomeres shorten during ageing of human fibroblasts.</a> Nature. 345, 458-460 (1990).</li><li>Hayflick, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limited+in+vitro+Lifetime+of+Human+Diploid+Cell+Strains.">The Limited in vitro Lifetime of Human Diploid Cell Strains.</a> Exp Cell Res. 37, 614-636 (1965).</li><li>Blackburn, 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=Telomeres:+no+end+in+sight.">Telomeres: no end in sight.</a> Cell. 77, 621-623 (1994).</li><li>Kim, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+association+of+human+telomerase+activity+with+immortal+cells+and+cancer.">Specific association of human telomerase activity with immortal cells and cancer.</a> Science. 266, 2011-2015 (1994).</li><li>Bodnar, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+life-span+by+introduction+of+telomerase+into+normal+human+cells.">Extension of life-span by introduction of telomerase into normal human cells.</a> Science. 279, 349-352 (1998).</li><li>Greider, C. W., Blackburn, 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=The+telomere+terminal+transferase+of+Tetrahymena+is+a+ribonucleoprotein+enzyme+with+two+kinds+of+primer+specificity.">The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity.</a> Cell. 51, 887-898 (1987).</li><li>Greider, C. W., Blackburn, 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=A+telomeric+sequence+in+the+RNA+of+Tetrahymena+telomerase+required+for+telomere+repeat+synthesis.">A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.</a> Nature. 337, 331-337 (1989).</li><li>Jacobs, S. A., Podell, E. R., Cech, T. R. <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+essential+N-terminal+domain+of+telomerase+reverse+transcriptase.">Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase.</a> Nat Struct Mol Biol. 13, 218-225 (2006).</li><li>Rouda, S., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+RNA-binding+domain+of+telomerase:+implications+for+RNA+recognition+and+binding.">Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding.</a> Structure. 15, 1403-1412 (2007).</li><li>Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H., Skordalakes, E. <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+telomerase+catalytic+subunit+TERT+binding+to+RNA+template+and+telomeric+DNA.">Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA.</a> Nat Struct Mol Biol. 17, 513-518 (2010).</li></ol>

这里介绍的方法可以为大量生产的T 的催化亚基谷盗可溶性活性形式，结构和生化研究端粒酶叔。 TC叔过度表达的方法是敏感微妙的变化，在温度或从上述这些细胞密度和蛋白表达水平可以显着地影响。具体来说，我们发现，诱导蛋白表达的细胞之前，在600 nm的光密度达到0.5，或降低温度至30 ° C诱导前，在蛋白质产量显着减少。 在净化方面，TC叔蛋...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>产品编号</th></tr><tr><td>罗塞塔（DE3）pLysS中细胞</td><td> Novagen公司</td><td> 70956 </td></tr><tr><td> 2YT肉汤</td><td> Teknova </td><td> Y0215 </td></tr><tr><td>经IPTG </td><td>黄金生物技术</td><td> I2481C </td></tr><tr><td> MISONIX Sonicator 3000 </td><td> Qsonica，LLC。 </td><td></td></tr><tr><td> ÄKTA净化FPLC </td><td> GE生命科学</td><td></td></tr><tr><td> NI - NTA Superflow树脂</td><td> Qiagen公司</td><td> 30410 </td></tr><tr><td> Amicon超- 15离心过滤装置</td><td> Millipore公司</td><td> UFC903008 </td></tr><tr><td> POROS 50 HS强阳离子交换包装</td><td>美国应用生物系统公司</td><td> 1-3359-06 </td></tr><tr><td> POROS 50 HQ强阳离子交换包装</td><td>美国应用生物系统公司</td><td> 1-2559-06 </td></tr><tr><td> Superdex 200 10/300体积排阻柱</td><td> GE生命科学</td><td> 17-5175-01 </td></tr><tr><td>酚：氯仿：异戊10MM三，pH值8，为1mM EDTA的25:24:1 </td><td>西格玛</td><td> P3803 - 100ML </td></tr><tr><td> RNaseZap </td><td> Ambion公司</td><td> AM9780 </td></tr><tr><td>重组RNasin核糖核酸酶抑制剂</td><td> Promega公司</td><td> N251B </td></tr><tr><td>的RNeasy迷你包</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td> DNA寡核苷酸</td><td>综合DNA技术</td><td></td></tr></tbody></table>

in vitro reconstitution of the active t. castaneum telomerase

努力找出端粒酶的催化亚基，叔，足够数量结构研究，已会见了十余有限的成功。在这里，我们目前的重组赤拟谷盗叔（TC叔）的分离和重组的积极 T.方法谷盗端粒酶的核糖核蛋白（RNP）复合物在体外 。 端粒酶是一个专门的反转录酶1，增加了短的DNA重复序列，称为端粒，线性染色体 2，有利于保护他们从终端到终端的融合和退化的3&#39;端。短段DNA复制，失去了在3号染色体的结束，如果没有端粒酶，细胞继续分裂，直至最终达到他们海弗利克极限4。此外，端粒酶在大多数体细胞在成人的 5中处于休眠状态，但活跃于癌细胞，促进细胞不死7。 最小的端粒酶由两个核心部分组成：蛋白亚基（叔），其中包括酶的催化亚基和一个完整的RNA组分（TER），其中包含的模板叔使用合成端粒8,9。在2008年之前，只有个别端粒酶域的结构已经解决了10,11。在这一领域的一个重大突破来自活跃的 12晶体结构测定，T的催化亚基谷盗端粒酶，TC叔1。 在这里，我们目前生产大量的活性，可溶性TC叔结构和生化研究，并重组端粒酶活性检测端粒酶在体外的RNP复杂的方法。使用的实验方法概述如图1所示。

Watch this Scientific Journal Video about In vitro Reconstitution of the Active T. castaneum Telomerase at JoVE.com

In vitro Reconstitution of the Active T. castaneum Telomerase

努力找出端粒酶的催化亚基，叔，足够数量结构研究，已会见了十余有限的成功。在这里，我们目前的重组赤拟谷盗叔隔离的方法（<em&gt; TC</em&gt;叔）和重组活动<em&gt; T。谷盗</em&gt;端粒酶的核糖核蛋白（RNP）复合物<em&gt;在体外</em&gt;。

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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In vitro Reconstitution of the Active T. castaneum Telomerase

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