A Chaperonin/Osmolyte Protein Folding Screen - STTR Phase I

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.