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Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane

Company: EMD Millipore
Catalog#: UFC903096
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Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae
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Date:
2017-10-05
[Abstract]  Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of Hsp90-Cdc37-Cdk4 kinase protein complex from Saccharomyces cerevisiae utilizing the viral 2A sequences to titrate the three proteins at similar levels. [摘要]  Hsp90,其伴侣伴侣Cdc37和激酶之间的相互作用已经在三十多年的生物化学研究中被证明在酵母与人类的生物体内在功能上是重要的。 然而,形成一个稳定的结构研究复合物是难以捉摸的。 在该方案中,我们描述了利用病毒2A序列以相似水平滴定三种蛋白质的来自酿酒酵母的Hsp90-Cdc37-Cdk4激酶蛋白复合物的表达和纯化。
【背景】Hsp90分子伴侣与其客体激酶之间的稳定形成复合物已经被证明在体外是难治性的。以前的工作表明,Hsp90的共伴伴Cdc37与昆虫Sf9细胞中的客体激酶的过表达导致Sf9 Hsp90,外源Cdc37和外源激酶(Vaughan等人,2006)之间的稳定复合物。然而,昆虫细胞培养需要特殊的设备,比其他研究较好的表达系统(如细菌和酵母)要难以进行遗传操作,并且显着较慢地生长和克隆。上述蛋白质在E中的共表达。大肠杆菌不产生可溶性激酶/稳定复合物。我们认为,酿酒酵母将具有必要的机制来帮助折叠和促进复合物的形成,并试图通过共同表达这些蛋白质来产生人Hsp90β,人Cdc37和人Cdk4激酶之间的复合物, S上。酵母。为了获得三种蛋白质的化学计量表达,我们利用病毒2A肽,其允许三个蛋白质在一个mRNA上转录,随后在翻译阶段切割。该系统已被用于人类细胞系和兔网状细胞(Kim等人,2011; ...

Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies
Author:
Date:
2017-02-05
[Abstract]  GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally containing a conserved GRAS domain, which is about 350-450 resides and can be subdivided into five distinct motifs with their name derived from the most prominent amino acids: LRI (leucine-rich region I), VHIID, LRII (leucine-rich region II), PFYRE and SAW and mainly function in the interaction between GRAS proteins and their partners (Sun et al., 2012).By phylogenetic analysis, the GRAS family can be divided into more than ten subfamilies, of which SCL4/7 is one important subgroup ... [摘要]  以前3个成员GAI,RGA和SRC命名的GRAS蛋白质已被发现在294个胚胎种,并由1,035个序列表示。它们属于植物特异性蛋白质家族,在植物生长和发育中起重要作用。该家族中的蛋白质被定义为最低限度地含有保守的GRAS结构域,其约为350-450个,并且可以细分为五个不同的基序,其名称源自最突出的氨基酸:LRI(富含亮氨酸的区域I),VHIID ,LRII(富含亮氨酸的区域II),PFYRE和SAW,并且主要在GRAS蛋白质与其配偶体之间的相互作用中起作用(Sun等人,2012)。通过系统发育分析,GRAS家族可以分为十多个亚科,其中SCL4 / 7是一个重要的亚组,对应对环境压力有作用。这里我们描述了Os-SCL7(水稻SCL4 / 7成员)的GRAS结构域的表达和纯化的详细方案,使我们能够使其结晶并确定其结构。

背景 GRAS蛋白是一个大家族,在植物发育和信号转导中起重要作用。结果表明,一些家庭成员如DELLAs起到GA响应植物生长的抑制作用,是GA信号通路(Murase等人,2008)中的关键调控目标,NSP1和NSP2起重要作用在调节结瘤发育和信号传导(Kaló等人,2005)中,蛋白质SCR和SHR一起在控制根和芽的径向图案中起重要作用(Helariutta et ...

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