Recombinant protein production in high cell density fermentation of R.eutropha using T7 polymerase system: Organophospho compounds are known to include several highly potent cholinesterase inhibitors. Such compounds can be readily obtained by conventional organic synthesis and their deployment can pose a serious threat to human life. Efforts to protect against the toxic effects of organophospho compounds have focused on enzymes that are able to hydrolyze the phosphoester bond and thereby substantially reduce their toxicity. The main obstacle in obtaining more than experimental quantities of this important chemical catalyst has been the unavailability of cost-effective enzyme expression system. GlycoFi is interested in developing genetic tools to overexpress proteins in high cell density fermentation. We propose to establish a T7 RNA polymerase based protein production system in a robust fermentation organism, Ralstonia eutropha. Our academic collaborators (Dr.Gerngross' lab at Dartmouth College) have been able to establish high cell densities of about 180g/L in R.eutropha under industrial feed conditions. T7 RNA polymerase system has been one of the most efficient recombinant protein production system developed. Although high yields (about 50% of total protein is recombinant protein) have been obtained in other bacteria (E.coli and Pseudomonas) in laboratory scales, various factors like proteolysis, inclusion body formation, difficult large scale fermentations have limited their production capabilities. In Phase I of the project, we would establish the T7 RNA polymerase system in R.eutropha and construct a plasmid to overexpress the gene of interest under the T7 promoter. Assuming that we are successful in achieving relative protein yields similar to other bacterial systems in R.eutropha, the high cell density fermentation capability of the organism with T7 expresssion system would enable us to produce recombinant protein titers of about 40-50 g/L. Successful completion of phase I is anticipated to demonstrate the superiority of the proposed bacterial expression system over existing methods of recombinant protein production. We would establish a low cost protein production system to produce high yields of Organophospho hydrolase, an enzyme used to reduce the toxicity of organophospho compounds (cholinesterase inhibitors). While we use OPH as a model enzyme, other applications that require large quantities of protein such as chemical/biological decontamination, bio-organic synthesis, materials for tissue engineering and molecular motors based on proteins, would greatly benefit from the new low cost protein production system.
Keywords: High Cell Density Fermentation, Organophosphohydrolase, Low Cost Protein Production
