A Novel Method for Producing Transgenic Enzymes and Peptides
Final rept. 1 Jun 2000-30 Nov 2003
DARTMOUTH COLL HANOVER NH OFFICE OF GRANTS AND CONTRACTS
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See report. We have successfully developed a novel a protein expression system based on the bacterium Raistonia eutropha. This system has been developed in our laboratory to overcome some of the shortcomings associated with recombinant protein expression in other bacteria e.g. poor fermentation performance, inclusion body formation and proteolysis. High level expression of organophosphohydrolase OPH, an enzyme prone to inclusion body formation in Escherichia coli has been demonstrated. A proteomics approach identified the promoter of the pexp haP gene as a strong, inducible promoter. By creating a translational fusion between the phaP promoter and oph gene and introducing this fusion into the chromosome, OPH titers of greater than 1 gL were achieved in high cell density fermentations. Multiple copies of the Pexp phaPoph translational fusion were introduced into the chromosome to further increase expression. A proportional increase in OPH titer was found with increasing copy number. An OPH titer of approximately 4.3 gL was measured in high cell density fermentation using a strain containing three copies of Pexp phaPoph translational fusion This represents the highest titer reported to date for OPH approximately 30 times greater than previously reported expression levels. Results have reported in two publications Srinivasan et al. 2003 Srinivasan et al., 2002.