NOVEL SOLUBILITY-ENHANCING PROTEIN EXPRESSION TECHNOLOGY

DESCRIPTION (provided by applicant): In the post-genomic era, a vast collection of protein-coding sequences is available for structural and functional analysis. Current structural genomics efforts seek to determine the complete repertoire of protein structures found in nature. The success of these efforts is expected to have a major impact on biomedicine, since most pharmaceuticals act upon proteins, and proteins are increasingly being developed for therapeutic uses. The favored host for recombinant protein expression is Escherichia coli. Despite many improvements, producing soluble proteins in E. coli is still a major bottleneck for structural genomics: typically, more than 50% of recombinant proteins are expressed in an insoluble form. Methods for screening growth conditions, host strains, and solubility enhancing fusion partners in the effort to optimize soluble protein expression are labor- and reagent-intensive. The goal of the proposed research is to develop a system of expression vectors and host strains to improve soluble expression of recombinant proteins. The system will include a yellow fluorescent protein tag that will function as an in vivo reporter of recombinant protein expression and solubility. This simple visual readout will facilitate expression screening efforts. Further, we will exploit this reporter capability to conduct genetic screens to identify novel protein fusion partners that promote soluble expression of difficult targets, and we will incorporate these solubility tags into products for protein expression and purification. The resulting system will enable parallel screening of fusion partners, host strains, and expression conditions to optimize target protein expression using a minimum amount of labor and reagents compared to conventional methods. PUBLIC HEALTH RELEVANCE: Genome sequencing efforts have provided a glimpse at the complete collection of proteins encoded by humans as well as many pathogens. Full realization of the potential biomedical benefits of this genomic information will require a more complete understanding of the structure and function of these proteins. Current structural genomics efforts seek to determine the complete repertoire of protein structures found in nature and revealed by genomic sequencing efforts. The success of these efforts is expected to have a major impact on biomedicine, since most pharmaceuticals act upon proteins, and proteins are increasingly being developed for therapeutic uses. Expression of proteins for structural and functional studies is usually undertaken in the bacterial host E. coli, but the production of foreign proteins in bacteria is hampered by an inability of many proteins to fold into a soluble conformation. The goal of the current proposed research is to develop a system for enhanced success of soluble protein expression in E. coli.