适应重组细菌性酸酶用于小GTPases的核酸交换
Peter H Frank1, Min Hong1, Brianna Higgins1
1NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
Protein expression and purification
|February 23, 2024
概括
研究人员开发了一种替代CIP-agarose用于RAS蛋白中的核酸交换的替代品. 结合珠子的细菌性酸酶 (BAP) 有效地用GTP类似物取代GDP,帮助癌症研究.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 癌症研究 癌症研究
背景情况:
- 鼠肉瘤病毒蛋白 (RAS) 是细胞生长的关键调节者,涉及到20-30%的人类癌症.
- RAS蛋白在活性 (GTP结合) 和非活性 (GDP结合) 状态之间循环,需要核酸交换来研究活性形式.
研究的目的:
- 开发一种替代商业上无法获得的CIP-agarose (牛犊肠性酸酶与阿加罗斯珠结合的替代品.
- 使用可访问的实验室材料建立一种可靠的RAS蛋白中核酸交换方法.
主要方法:
- 产生具有亲和度标签的细菌性酸酶 (BAP).
- 将BAP固定在阿加罗斯珠上.
- 在核酸交换协议中验证BAP-糖合物.
主要成果:
- 开发的BAP - 糖结合物有效地促进了RAS蛋白中的完整核酸交换.
- 这种方法为标准的CIP-agarose协议提供了可行的替代方案.
- 该协议使用了一般可用的实验室设备.
结论:
- 细菌性酸酶 (BAP) 固定在阿加罗斯珠上,可以作为CIP-agarose的有效替代品.
- 这种替代方法解决了供应链问题,并支持在癌症中继续研究RAS蛋白.
相关概念视频
GTPases and their Regulation
8.4K
Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒ small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins,...
Large G-proteins,...
8.4K
Small GTPases - Ras and Rho
4.0K
Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
Three regulatory proteins control their activity:
4.0K
Activation and Inactivation of G Proteins
7.2K
Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high...
7.2K
Coat Assembly and GTPases
3.5K
Vesicles incorporate different coat protein subunits in different cell locations, which changes the properties of the coat, such as the shape and geometry of the transport vesicles. Thus, vesicle coat proteins also play a significant role in cargo selection.
Coat assembly depends on the local availability of phosphatidylinositol phosphates or PIPs and GTP-binding proteins. Adaptor proteins, which link the coat proteins to the membrane, bind to these PIPs and play a crucial role in controlling...
Coat assembly depends on the local availability of phosphatidylinositol phosphates or PIPs and GTP-binding proteins. Adaptor proteins, which link the coat proteins to the membrane, bind to these PIPs and play a crucial role in controlling...
3.5K
Rab Proteins
3.9K
Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
3.9K


