Research Scientist - Engineer Senior
CurrentImplement scaled up fermentation and purification processes for Paraoxanase (PON) family members expressed in E.coli Host. This family exhibits the potential to combat exposure to OrganoPhosphate nerve agents (OPNA's). My research is focused on increasing soluble active PON1 levels found in cell lysates, as well as evaluating the possibility of refolding active enzyme from Inclusion bodies. Initial scale up culture efforts have established Glucose Fed-batch and Batch fermentation protocols that have expanded the scale of culture leading to high levels of insoluble PON1 in inclusion bodies, and low levels of soluble active PON1 from the cell lysates, similar to levels exhibited at shaker flask scale. A confounding technical challenge stems from the active enzyme requirement for calcium. Fermentation of E.coli to high density relies upon significant amounts of phosphate in the culture media for buffering and nucleic acid synthesis in the expanding culture. The usual levels of phosphate utilized to generate high density E.coli cultures conflict with calcium solubility at levels required to stabilize the active enzyme. Part of my current focus is on adapting expression constructs to culture methods targeting expression to the periplasmic space, putting the gene behind the pHoA promotor using the pHoA secretion leader so that low phosphate culture methods can be utilized with sufficient calcium to increase soluble PON1 levels. Further enhancement may be obtained through combining periplasmic expression with "leaky" host cells, or u constructs targeting secretion of periplasmic expressed proteins into the culture media. i have also established culture methods utilizing the carbon based metabolism method of auto induction in E.coli. which have increased soluble enzyme production 3-6 times volumetrically at 2 times the final biomass. This is a small overall increase of soluble activity, but the data suggest the quantity can be biased by culture and induction methods.
