Novel fluorescent protein expression vector that simplifies the solubilization of membrane proteins

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ABSTRACT Simple protein mutations can have serious consequences by causing diseases that affect people's lives. Protein therapeutics are developed to target and replace those protein mutations, and essentially to heal the disease. Distinctively, membrane proteins make up over 60% of today's drug targets. However, cumbersome, time- consuming, and often ineffectual purification methods have hampered membrane protein purification and downstream experimentation. The long-term objective of this application is to develop a rapid and simplified method for enhanced expression and tailoring purification to each specific membrane protein; this would greatly enhance commercial opportunities for this important therapeutic sector. The baculovirus expression vector system (BEVS) is commonly used for production of proteins for structure- function studies, vaccines and therapeutics, but membrane proteins are often poorly expressed and difficult to purify. ParaTechs (www.ParaTechs.com) currently markets insect cells and vectors that provide heightened expression of foreign genes due to delayed lysis and increased overall health of infected cells. This proposal seeks to expand our baculovirus product line through the development of this innovative tool by combining the vankyrin-enhanced baculovirus expression vector system with a novel fluorescent fusion tag that will aid the expression and solubilization of membrane proteins. Aim one is to design baculovirus transfer vectors and viruses harboring both the vankyrin gene and membrane proteins fused to the novel fluorescent tag. The second aim will evaluate the stability of the fluorescent tag in the detergents selected for the solubilization screen. Aim three of this application will compare the efficiency and efficacy of membrane protein expression and purification using a new solubilization screen that is compatible with the recombinant baculoviruses produced in the first aim. The here developed fluorescent selection technology will allow enhanced functional protein production, rapid identification of detergents that enable successful solubilization, and easy optimization of protein purification conditions. This innovative technology will overcome two major barriers in the development of protein therapeutics, namely the expression of large quantities of pure, properly processed membrane proteins and to solubilize active proteins from membranes.