This Small Business Innovation Research (SBIR) Phase I project proposes a new expression platform in Escherichia coli applicable to both drug discovery and biopharmaceutical manufacturing. It solves and leverages recombinant protein misfolding in E. coli. Small molecular weight compounds are used in conjunction with high recombinant protein expression in E. coli to produce proteins with their native structure. In the area of drug discovery this allows one to obtain properly-folded proteins for structural and functional genomics, high-throughput screening, and to identify specific protein stabilizers for protein-misfolding diseases. For biopharmaceutical manufacturing, recombinant protein inclusion body formation is prevented and refolding steps are eliminated. Cultures of E. coli containing a plasmid encoding a gene for the recombinant protein of interest are allowed to grow to a desired cell density and then an innovative condition-dependent change is made that allows the passive influx of small molecular weight compounds into the E. coli cytoplasm during protein synthesis that support proper protein folding. This proposal will examine the feasibility of this condition-dependent change and the alteration of the cell?s cytoplasm in a prescribed way to produce properly-folded proteins by monitoring the soluble and functional expression of selected recombinant proteins. The broader impact/commercial potential of this project will be a gene expression platform that is an enabling technology that has applications wherever properly-folded recombinant proteins are utilized. The described system can produce recombinant proteins in E. coli that would otherwise be toxic such as membrane proteins, which are highly desirable drug targets but are recalcitrant proteins for functional and structural genomics research. It is the first E. coli system described that can be leveraged to screen for drugs directed against protein-misfolding diseases. It is a new avenue for identifying drugs to treat devastating and costly diseases including p53-mediated cancers, Alzheimer's, Parkinson's, Tay-Sachs, and amyotrophic lateral sclerosis. The proposed expression platform can potentially make these proteins more research accessible. Inclusion body formation in E. coli may also benefit from this technology. This is particularly applicable to recombinant proteins intended as biopharmaceuticals. Preventing inclusion bodies increases process yields, shortens process development timelines and reduces commercial manufacturing costs. Hence, this proposal describes a new tool to discover drugs for unmet medical needs, expedite drug discovery, and streamline biopharmaceutical development and manufacturing; all of which will ultimately provide economical and societal benefits.
