HIGH THROUGHPUT CLONING OVEREXPRESSION AND PURIFICATION OF ACTIVE MEMBRANE PROT

DESCRIPTION (provided by applicant): Functional analysis of proteins is essential to thoroughly understand the basic interactions essential for life. However, obtaining pure proteins for basic research and drug screening is limited by technical bottlenecks. For example, high throughput studies are severely hampered by difficulties in producing and detecting active proteins, especially those that are bound to cellular membranes. Only a small fraction of known proteins have been thoroughly characterized. Of these the vast majority are soluble proteins. Although membrane proteins may constitute a third of the genes in an organism, they represent only 0.5% of the 3D protein structures that have been deciphered. We propose a system to allow robust production and isolation of proteins in a soluble, fully functional form. It will be particularly well-suited to analysis of membrane proteins, and it will be amenable to screening for active membrane proteins in a large-scale format. We plan to make use of novel cloning vectors that stably maintain otherwise "unclonable" genes. A new E. coli cell line will be developed that produces recombinant membrane proteins in easily-purified vesicles. This system will also incorporate a new affinity capture reagent that yields active, undenatured protein with 95% purity. In Phase II, we plan to extend this technology to facilitate expression of the most difficult proteins. Novel expression vectors, cell lines, and purification reagents will be developed into individual products and complete kits. These products will enable researchers to study a wide variety of soluble or membrane- bound proteins that otherwise would be difficult or impossible to produce. This technology will have nearly universal applications in biotechnology, including critical importance in pharmaceutical research (antimicrobials, immunity, inflammation) and environmental research (biofilms, microbial energy production). An improved understanding of the structure and function of membrane proteins in prokaryotes and eukaryotes could result in significant benefits for human and animal health. For example, the ability to express pharmaceutically relevant membrane proteins, such as ion channels and G protein-coupled receptors, in an active state could lead to new therapies for numerous diseases. Many cancer therapeutics become ineffective due to the emergence of multidrug efflux proteins in the cell membrane, and an improved understanding of these proteins could result in improved cancer therapies.