LUCIGEN CORPORATION — Department of Health and Human Services SBIR Phase II: DESCRIPTION (provided by applicant): A major goal in the post-genomic era is to express th

Description

DESCRIPTION (provided by applicant): A major goal in the post-genomic era is to express the vast collection of protein- coding sequences, eventually resulting in a better understanding of protein interactions and the development of novel therapeutics. Themost favored host for heterologous recombinant protein expression is Escherichia coli. Despite many improvements, producing soluble proteins in E. coli is still a major bottleneck for structural genomics: typically, gt50% of recombinant proteins are expressed in an insoluble form. Methods to optimize soluble protein expression are labor- and reagent-intensive. They involve screening for growth conditions, host strains, and solubility enhancing fusion partners and assessing solubility by cell lysis, fractionation and gel electrophoresis. The goal of the proposed research is to develop an integrated system of expression vectors and host strains to improve soluble expression of recombinant proteins. The system will include a novel yellow fluorescent protein tagthat will function as an in vivo reporter of expression and solubility of the recombinant protein. This simple visual readout will facilitate individual and high-throughput expression screening. Further, we will exploit this reporter system to conduct genetic screens for novel protein fusion partners that promote soluble expression of difficult targets. We will validate the resulting solubility tags and incorporate them into a suite of products for protein expression and purification. The resulting system will enable high-throughput optimization of soluble protein expression through parallel screening of fusion partners, host strains, and expression conditions. It will likewise be a great advantage for expression of individual proteins, minimizing the use of labor and reagents. The success of these efforts is expected to have a major impact on biomedicine, both for academic purposes and for development of protein therapeutics. PUBLIC HEALTH RELEVANCE: Expression of proteins for structural and functional studies is usually undertaken in the bacterial host Escherichia coli. Importantly, 30% of the 151 biopharmaceutical proteins, worth 53B in product sales in 2005, were produced in E. coli. However, production of foreign proteins in bacteria is hampered by the inability of many proteins to fold into a soluble conformation. The goal of the current research proposal is to develop a system for enhanced expression of soluble protein in E. coli.