A Chaperonin/Osmolyte Protein Folding Screen - STTR Phase I

伴侣蛋白/渗透调节蛋白折叠筛选 - STTR 第一阶段

• 批准号: 7221111
• 负责人: Mark T Fisher
• 金额: $ 10万
• 依托单位: EDGE BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7221111
• 关键词: Chaperonin; Osmolyte; Protein; Folding; Screen

DESCRIPTION (provided by applicant): One of the largest bottlenecks in high-throughput protein structure determination (structural genomics) and in subsequent drug targeting (pharmocogenomics) is the acquisition of soluble correctly folded functional protein products. Unfortunately, a large amount of the protein expression systems that are commonly used yield insoluble or soluble misfolded proteins. We have developed a new folding process that employs a combination of bacterial GroEL and naturally occurring cellular osmolytes. This system is successful at folding a number of proteins that cannot fold from either the chaperonin or osmolytes alone. The chaperonin/osmolyte folding system is synergistic and folds proteins to high yields, at high concentrations, and at physiological temperatures. Briefly, denatured proteins are captured by the chaperonin as a stable partially folded intermediate that can be concentrated and immobilized. In this trapped form, the captured folding intermediates can be released from the chaperonin using osmolyte solutions or mixtures of osmolytes. This system can successfully fold 1) aggregation prone proteins, 2) insoluble proteins isolated from inclusion bodies, 3) misfolded soluble proteins, and 4) misfolded proteins found in disease states. Studies have shown that osmolytes can be segregated in categories of folding osmolytes and anti-aggregation osmolytes and we have shown that the latter can enhance the efficiency of the system by partially stabilizing denatured proteins prior to complexing with GroEL. In this proposal, our first specific aim outlines our efforts to demonstrate that the GroEL/osmolyte process can be streamlined to allow researchers to fold proteins from inclusion bodies in a single pot reaction. An important milestone in these studies will be the demonstration that the model proteins can be crystallized, an important and demanding endpoint. These studies will use dimeric protein ?5-3-ketosteriod isomerase and human mitochondrial phosphoenolpyruvate carboxykinase. The former is a well characterized and crystallized protein; the latter is not. In the second aim, we will assess the extent to which serum and tissue proteins in general will serve as substrates for this process. Using serum and tissue Iysates, we intend to show that a broad spectrum of proteins that do not fold without extensive manipulation in vitro will do so quickly and efficiently with this system. Our third aim is to streamline the purification process for GroEL so that the costs of the protein which are currently very high, will not limit the usefulness of the system to the research community.

描述（由申请人提供）：高通量蛋白质结构测定（结构基因组学）和后续药物靶向（药物基因组学）的最大瓶颈之一是获取可溶性正确折叠的功能蛋白质产品。不幸的是，大量常用的蛋白质表达系统产生不溶性或可溶性错误折叠蛋白质。我们开发了一种新的折叠工艺，该工艺结合了细菌 GroEL 和天然细胞渗透剂。该系统成功地折叠了许多不能单独由伴侣蛋白或渗透调节剂折叠的蛋白质。伴侣蛋白/渗透剂折叠系统具有协同作用，可在高浓度和生理温度下以高产率折叠蛋白质。简而言之，变性蛋白质被伴侣蛋白捕获，作为可以浓缩和固定的稳定的部分折叠中间体。在这种捕获形式中，捕获的折叠中间体可以使用渗透剂溶液或渗透剂混合物从伴侣蛋白中释放。该系统可以成功折叠 1) 易于聚集的蛋白质，2) 从包涵体中分离出的不溶性蛋白质，3) 错误折叠的可溶性蛋白质，以及 4) 在疾病状态下发现的错误折叠的蛋白质。研究表明，渗透剂可以分为折叠渗透剂和抗聚集渗透剂，我们已经证明后者可以在与 GroEL 复合之前通过部分稳定变性蛋白质来提高系统的效率。在本提案中，我们的第一个具体目标概述了我们努力证明 GroEL/渗透剂过程可以简化，以允许研究人员在单锅反应中折叠来自包涵体的蛋白质。这些研究的一个重要里程碑将是证明模型蛋白质可以结晶，这是一个重要且要求严格的终点。这些研究将使用二聚体蛋白α5-3-酮类异构酶和人线粒体磷酸烯醇丙酮酸羧激酶。前者是一种经过充分表征和结晶的蛋白质；后者则不然。在第二个目标中，我们将评估血清和组织蛋白一般在多大程度上充当该过程的底物。使用血清和组织裂解物，我们打算证明，未经体外大量操作就不会折叠的广谱蛋白质将通过该系统快速有效地折叠。我们的第三个目标是简化 GroEL 的纯化过程，以便目前非常高的蛋白质成本不会限制该系统对研究界的有用性。

项目成果

GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore.

• DOI: 10.1038/nsmb.1442
• 发表时间: 2008-07
• 期刊: Nature structural & molecular biology
• 影响因子: 16.8

Identifying protein stabilizing ligands using GroEL.

使用 GroEL 识别蛋白质稳定配体。

• DOI: 10.1002/bip.21319
• 发表时间: 2010
• 期刊: Biopolymers
• 影响因子: 2.9
• 作者: Naik,Subhashchandra;Haque,Inamul;Degner,Nick;Kornilayev,Boris;Bomhoff,Gregory;Hodges,Jacob;Khorassani,Ara-Azad;Katayama,Hiroo;Morris,Jill;Kelly,Jeffery;Seed,John;Fisher,MarkT
• 通讯作者: Fisher,MarkT