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.
This Small Business Innovation Research Phase I project proposed a new Escherichia coli expression platform for properly-folded recombinant proteins that is anticipated to have potential application towards drug discovery and biopharmaceutical manufacturing. Drug discovery efforts would be facilitated by the use of properly-folded proteins for structural and functional genomics, high-throughput screening, and identifying specific protein stabilizers for protein-misfolding diseases. Biopharmaceutical manufacturing would benefit by avoiding insoluble inclusion body formation and the elimination of costly protein refolding processes. The sponsored work investigated the ability of small molecular weight compounds to access the bacterial cytoplasm during protein synthesis in response to an innovative condition-dependent change and subsequently to stabilize the folding polypeptide chain during recombinant protein production. When growing cultures of E. coli containing the gene for a specific target protein that is produced in a misfolded state were exposed to the condition-dependent change followed by treatment with a library of small molecular weight compounds, many of these compounds gained access to the bacterial cytoplasm and rescued the target protein in a properly-folded state. The proper folding of the target protein was evidenced by an increase in its functional activity. Approximately 41% of the small molecular weight compounds tested rescued the functional activity of the target protein reproducibly in at least 3 different experiments performed on separate days. The maximum increase in properly-folded target protein by any small molecular weight compound approached 700%. The results of this study demonstrate the following: 1) the innovative condition-dependent change does allow small molecular weight compounds to access the bacterial cytoplasm and 2) certain small molecular weight compounds can rescue properly-folded recombinant proteins that would otherwise be misfolded and insoluble. The former finding opens a new investigative field into altering the E. coli cytoplasm for beneficial purposes and the latter finding leads to a pathway for developing a new recombinant expression platform in E. coli that has multiple potential applications in biomedical research, development, and commercialization.
