A Novel Method for Producing Transgenic Enzymes and Peptides

Abstract

Public Reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comment regarding this burden estimates or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for information Operations and Reports, 1215 The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision, unless so designated by other documentation. We have successfully developed a novel a protein expression system based on the bacterium Ralstonia eutropha. This system has been developed in our laboratory to overcome some of the shortcomings associated W\\.h 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 Eschehchia colt has been demonstrated. A proteomics approach identified the promoter of the phaP 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 g/L were achieved in high cell density fermentations. Multiple copies of the Pphap'.'-oph 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 g/L was measured in high cell density fermentation using a strain containing three copies of Pphap'-'oph 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; Srinivasanetal., 2002 REPORT DOCUMENTATION PAGE (SF298) (Continuation Sheet)Scientific progress and accomplishments:Introduction:With advances in protein engineering, there has been a surge in the number of newly discovered proteins and protein function as well as the ability to engineer the properties of such proteins. Enhancement of protein production is a primary goal of recombinant microbial process development. This can be achieved by increasing the amount of recombinant protein per cell (specific productivity) and/or increasing the cell concentration per unit time (cell productivity). In order to improve volumetric productivity in a cost effective manner, recombinant proteins are often produced in high cell density fermentations. High cell density fermentation has many advantages over normal fermentations in that they have a higher final product concentration, reduced waste water and higher overall productivity and hence lower setup and...

Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms

“…Like enzymatic glycosylation, this strategy typically leads to more homogeneous glycoforms than those produced naturally by CHO. P. pastoris was the first host to be successfully engineered to generate homogeneous glycoforms on antibodies that incorporate all five sugars (Gerngross, ; Jacobs, Geysens, Vervecken, Contreras, & Callewaert, ). The same approach has been applied successfully to other yeast and fungi (Figure b).…”