Engineering E. coli for periplasmic protein production
Small Business Information
SCARAB GENOMICS, LLC, 1547 JEFFERSON ST, MADISON, WI, 53711
AbstractDESCRIPTION (provided by applicant): Escherichia coli has numerous advantages as a host for the manufacture of recombinant proteins. So far, with a few notable exceptions, it has proven difficult to make disulfide bonded proteins in this host. The few successes have been achieved mainly by exploiting the natural secretion machinery that enables proteins to be exported to the periplasmic space, which supports correct processing, folding and disulfide bonding. We propose a novel approach to improving the periplasmic environment, by greatly reducing the number of native periplasmic proteins in order to facilitate purification and reduce the load on the secretory machinery, which is not only sensitive to overloading, but also prone to jamming by over-expressed recombinant proteins. This should greatly increase its capabilities for the commercial production of medically and economically important biopharmaceuticals, diagnostics, vaccines and proteins to support research in academic and industrial biomedical research. Genes for periplasmic proteins that are dispensable under industrial fermentation conditions will be deleted, preserving genes necessary for growth, metabolism and protein expression, secretion and processing. This project builds on the recent development by the PI and coworkers of a powerful 'starless' genome engineering technology and novel E. coli MDS strains, from which a large amount of unnecessary and undesirable horizontally transferred genomic material has already been deleted, without compromising growth and function under controlled conditions. MDS42 is free of mobile genetic elements that contribute substantially in the wild-type to genetic mutation and instability. Many genes closely related to virulence and pathogenecity determinants have also been eliminated. The project will also create many new strains of E. coli for research and will advance the science of large-scale genome engineering. In Phase I, feasibility will be established by investigating the expression, secretion into the periplasm, and processing of proteins in MDS strains to determine their suitability for the Phase II periplasmic re-engineering program. We will also evaluate the MDS strains for growth under industrially relevant fermentation conditions. An appropriate MDS starting strain will be selected for Phase II.
* information listed above is at the time of submission.