A Chaperonin/Osmolyte Protein Folding Screen - STTR Phase I

描述(申请人提供)：高通量蛋白质结构测定(结构基因组学)和随后的药物靶向(药物基因组学)的最大瓶颈之一是获得可溶的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了不能溶解或可溶的错误折叠的蛋白质。我们开发了一种新的折叠工艺，使用了细菌GroEL和自然产生的细胞渗透分子的组合。这个系统成功地折叠了许多蛋白质，这些蛋白质不能单独从伴侣或渗透压细胞折叠。伴侣蛋白/渗透压折叠系统是协同作用的，在高浓度和生理温度下折叠蛋白质以获得高产量。简而言之，变性蛋白被伴侣蛋白捕获，作为一种稳定的部分折叠的中间体，可以被浓缩和固定。在这种被捕获的形式中，捕获的折叠中间体可以使用渗透分子溶液或渗透分子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，渗透分子可以分为折叠渗透分子和反聚集渗透分子，我们已经证明，后者可以通过在与GroEL络合之前部分稳定变性蛋白来提高系统的效率。在这项提案中，我们的第一个具体目标概述了我们所做的努力，以证明GroEL/渗透分子过程可以被简化，使研究人员能够在单一的POT反应中折叠包涵体中的蛋白质。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，这是一个重要和苛刻的终点。这些研究将使用二聚体蛋白？5-3-酮类固醇异构酶和人线粒体磷酸烯醇式丙酮酸羧酸激酶。前者是一种具有良好特性和结晶的蛋白质；后者则不是。在第二个目标中，我们将评估血清和组织蛋白在多大程度上作为这一过程的底物。使用血清和组织裂解液，我们打算证明在体外没有广泛的操作就不会折叠的广泛的蛋白质将通过这个系统快速而有效地进行折叠。我们的第三个目标是简化GroEL的纯化过程，使目前非常高的蛋白质成本不会限制该系统对研究界的用途。

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.