A Chaperonin/Osmolyte Protein Folding Screen - STTR Phase I

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性正确折叠的功能蛋白质产物。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的蛋白质。我们已经开发了一种新的折叠过程，采用细菌GroEL和自然发生的细胞渗透物的组合。这个系统成功地折叠了许多单独使用伴侣蛋白或渗透蛋白都无法折叠的蛋白质。伴侣蛋白/渗透物折叠系统是协同的，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物，可以浓缩和固定。在这种被捕获的形式中，捕获的折叠中间体可以使用渗透液溶液或渗透液混合物从伴侣蛋白中释放出来。该系统可以成功折叠1)易聚集蛋白，2)从包涵体中分离的不溶性蛋白，3)错误折叠的可溶性蛋白，以及4)在疾病状态中发现的错误折叠蛋白。研究表明，渗透物可以分为折叠渗透物和反聚集渗透物，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的努力，以证明GroEL/渗透过程可以简化，使研究人员能够在单锅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可以结晶，这是一个重要而苛刻的终点。这些研究将使用二聚体蛋白？5-3-酮甾异构酶与人线粒体磷酸烯醇丙酮酸羧激酶。前者是一种特性良好的结晶蛋白；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将作为该过程底物的程度。使用血清和组织分析液，我们打算表明，在体外不进行大量操作的广谱蛋白质将在该系统下快速有效地折叠。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有用性。

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.