Research Project
8501542
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. The most 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, >50% 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 tag that 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.
