Name: quantitative fret (f&#246;rster resonance energy transfer) analysis for senp1 protease kinetics determination
Uploaded: 2013-02-21T12:00:00
Description: Watch this Scientific Journal Video about Quantitative FRET FÃƒÂ¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination at JoVE.com

我们非常感谢维克多GJ罗杰斯宝贵的意见。我们感谢所​​有的廖组的成员非常密切的合作工作，并帮助这项研究。这项研究是由美国国立卫生研究院（批准AI076504 JL）的支持。

1。质粒的构建。 <ol><li>通过PCR扩增的基因的开放阅读框架，PCR产物克隆到pCRII-TOPO载体中。 </li><li>通过测序确认产品，和克隆的cDNA编码CyPet-（预SUMO1）-YPet，-SUMO1 CyPet，YPet和催化结构域的SENP1到pET28（二）的N-末端六组氨酸标签的向量。 </li></ol> 2。蛋白质表达与纯化<ol><li>转化大肠杆菌菌株BL21（DE3）细胞与pET28（二）编码CyPet-（预SUMO1）-YPet，-SUMO1 CyPet，YPet和催化...

<ol>
	<li>Johnson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+modification+By+SUMO.">Protein modification By SUMO.</a> Annual Review of Biochemistry. 73, 355-382 (2004).</li><li>Gill, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO+and+ubiquitin+in+the+nucleus:+different+functions,+similar+mechanisms.">SUMO and ubiquitin in the nucleus: different functions, similar mechanisms.</a> Genes &amp; Development. 18, 2046-2059 (2004).</li><li>Hay, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO:+a+history+of+modification.">SUMO: a history of modification.</a> Molecular Cell. 18, 1-12 (2005).</li><li>M&#252;ller, S., Hoege, C., Pyrowolakis, G., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO,+ubiquitin's+mysterious+cousin.">SUMO, ubiquitin's mysterious cousin.</a> Nature. , 202-210 (2001).</li><li>Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SP-RING+for+SUMO:+new+functions+bloom+for+a+ubiquitin-like+protein.">SP-RING for SUMO: new functions bloom for a ubiquitin-like protein.</a> Cell. , 5-8 (2001).</li><li>Drag, M., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeSUMOylating+enzymes-SENPs.">DeSUMOylating enzymes-SENPs.</a> IUBMB Life. 60, 734-742 (2008).</li><li>Mukhopadhyay, D., Dasso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+in+reverse:+the+SUMO+proteases.">Modification in reverse: the SUMO proteases.</a> Trends in Biochemical Sciences. 32, 286-295 (2007).</li><li>Reverter, D., Lima, C. D. <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+SENP2+protease+interactions+with+SUMO+precursors+and+conjugated+substrates.">Structural basis for SENP2 protease interactions with SUMO precursors and conjugated substrates.</a> Nature Structural &amp; Molecular Biology. 13, 1060-1068 (2006).</li><li>Shen, L., 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=SUMO+protease+SENP1+induces+isomerization+of+the+scissile+peptide+bond.">SUMO protease SENP1 induces isomerization of the scissile peptide bond.</a> Nature Structural &amp; Molecular Biology. 13, 1069-1077 (2006).</li><li>Horton, R., Strachan, E., Vogel, K., Riddle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+substrate+for+deubiquitinating+enzymes+based+on+time-resolved+fluorescence+resonance+energy+transfer+between+terbium+and+yellow+fluorescent+protein.">A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein.</a> Analytical Biochemistry. 360, 138-143 (2007).</li><li>Mikolajczyk, J., 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=Small+Ubiquitin-related+Modifier+(SUMO)-specific+Proteases:+PROFILING+THE+SPECIFICITIES+AND+ACTIVITIES+OF+HUMAN+SENPs.">Small Ubiquitin-related Modifier (SUMO)-specific Proteases: PROFILING THE SPECIFICITIES AND ACTIVITIES OF HUMAN SENPs.</a> Journal of Biological Chemistry. 282, 26217-26224 (2007).</li><li>Kolli, N., Mikolajczyk, J., Drag, M., Mukhopadhyay, D., Moffatt, N., Dasso, M., Salvesen, G., Wilkinson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distrubition+and+paralogue+specificity+of+mammalian+deSUMOylating+enzymes.">Distrubition and paralogue specificity of mammalian deSUMOylating enzymes.</a> Biochem. J. , 335-344 (2010).</li><li>Drag, M., Mikolajczyk, J., Krishnakumar, I. M., Huang, Z., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+profiling+of+human+deSUMOylating+enzymes+(SENPs)+with+synthetic+substrates+suggests+an+unexpected+specificity+of+two+newly+characterized+members+of+the+family.">Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family.</a> Biochemical Journal. 409, 461 (2008).</li><li>Engels, I. H., 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+time-resolved+fluorescence+resonance+energy+transfer-based+assay+for+DEN1+peptidase+activity.">A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity.</a> Analytical Biochemistry. 390, 85-87 (2009).</li><li>Martin, S., Hattersley, N., Samuel, I., Hay, R., Tatham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-resonance-energy-transfer-based+protease+activity+assay+and+its+use+to+monitor+paralog-specific+small+ubiquitin-like+modifier+processing.">A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing.</a> Analytical Biochemistry. 363, 83-90 (2007).</li><li>Nguyen, A. W., Daugherty, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+optimization+of+fluorescent+proteins+for+intracellular+FRET.">Evolutionary optimization of fluorescent proteins for intracellular FRET.</a> Nature Biotechnology. 23, 355-360 (2005).</li></ol>

FRET技术用于研究前SUMO1的成熟，由SENP1 9。 CFP-YFP用作FRET对和比例的分析，这是受体捐助排放比，用于表征的动力学性质。然而，有没有考虑供体和受体在传统比例的自身荧光共振分析。比例不直接与衬底消化量。 在这里，我们报告一个高度敏感的基于FRET的的蛋白酶分析研究动力学的前SUMO1的成熟SENP1。对比的比例的方法，从根本上提高一个新的理论方法与共振信号?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> PCR II TOPO包</td><td> Invitrogen公司</td><td> K466040 </td></tr><tr><td>的2xYT </td><td>研究产品Internationa.l公司</td><td> X15600 </td></tr><tr><td>镍-NTA琼脂糖</td><td> Thermo Scientific的</td><td> 88222 </td></tr><tr><td>考加号（布拉德福德）含量摄政</td><td> Thermo Scientific的</td><td> 23238 </td></tr><tr><td> 384孔板（玻璃底） </td><td>格雷纳</td><td> 781892 </td></tr><tr><td> FlexStation II 384酶标仪</td><td>分子器件</td><td></td></tr></tbody></table>

quantitative fret (f&#246;rster resonance energy transfer) analysis for senp1 protease kinetics determination

可逆的翻译后修饰的蛋白质与泛素或泛蛋白 - 样的蛋白质（Ubls）被广泛地用于动态调节蛋白的活性，并具有在许多生物过程中的各种不同的角色。例如，SUMO共价键修改大量蛋白质的重要​​角色，在许多细胞过程，包括细胞周期调控，细胞的生存和死亡，DNA损伤反应和应激反应1-5。 SENP，SUMO特异性蛋白酶，成熟的SUMO前体肽链内切酶的功能，或作为isopeptidase删除SUMO从靶蛋白SUMO化循环刷新1,3,6,7。 或特异性的酶的催化效率最好是其特征在于由比率的动力学常数，K 猫 / K M。在几项研究中，的动力学参数的SUMO-SENP对已确定的各种方法，包括基于聚丙烯酰胺凝胶Western-blot检测，RADIOActive的标记底物，荧光化合物或蛋白质标记底物8-13。然而，聚丙烯酰胺凝胶为基础的技术，它使用“本土”的蛋白质，但费力，技术要求高，不容易借给自己详细的定量分析。所得到的K 猫 / K M从使用的四肽或蛋白的研究，与ACC（7 -氨基-4-carbamoylmetylcoumarin）或AMC（7 -氨基-4 -甲基香豆素）荧光团要么达两个数量级低于天然底物或不能明确区分和肽链内切酶的活动SENPs。 最近，基于FRET的蛋白酶检测使用，研究去泛素酶（DUBS）或SENPs的FRET对青色荧光蛋白（CFP），黄色荧光蛋白（YFP）9,10,14,15。受体发射的比率被用作供体发射的定量参数FRET蛋白酶交流信号监视器拉了一的决心。然而，这种方法忽略的信号交叉污染的受体和供体受体和供体荧光发射波长，因而是不准确的。 我们开发了一个新的高度基于FRET的蛋白酶敏感和定量分析，确定动力学参数的SUMO1成熟的SENP1。一种工程FRET对CyPet的和YPet显着提高的FRET效率和荧光量子产率，被用来生成的CyPet（预SUMO1）-YPet基板16。我们差异化和量化的绝对贡献的供体和受体和FRET在接受器和发射波长，荧光信号。得到的k 猫 / K M的值（3.2±0.55）×10 7 M -1 -1 SENP1朝向预SUMO1，这是与一般的酶动力学参数。因此，这种方法是有效的，并可以使用了一个通用的方法来描述以及其他蛋白酶。

Watch this Scientific Journal Video about Quantitative FRET FÃƒÂ¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination at JoVE.com

Quantitative FRET FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

涉及的FRET（福斯特共振能量转移）信号的定量分析酶动力学研究的一种新方法。<em&gt; K<sub&gt; M</sub</em&gt;和<em&gt; K<sub&gt;猫</sub</em&gt; SENP1（SUMO / Sentrin特定的蛋白酶1）预SUMO1（小泛素样修饰）的催化结构域的水解获得。这个定量的基于FRET的蛋白酶动力学研究的一般原则可以应用到其他的蛋白酶。

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

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Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

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