title: Production of recombinant proteins using multiple-copy gene integration in high-cell-density fermentations of Ralstonia eutropha.
abstract: We have previously reported the development of a novel protein expression system based on Ralstonia eutropha. In this study we report on the influence of gene copynumber on recombinant protein expression in R. eutropha. We compare recombinant gene stability and expression levels of chromosomal integration with a plasmid-based expression system. Single, double, and triple copies of a gene encoding organophosphohydrolase (OPH), an enzyme prone to inclusion-body formation in E. coli, were integrated into the R. eutropha chromosome. A linear increase between the concentration of soluble, active OPH and gene copynumber was found. Using a triple-copy integrant, we were able to produce approximately 4.3 g/L of OPH in a high-cell-density fermentation. This represents the highest titer reported to date for this enzyme, and is approximately 30 times greater than expression levels reported in E. coli.
authors.affiliation: Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, New Hampshire 03755, USA.
authors.forename: Sriram
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Gavin C
authors.initials: GC
authors.lastname: Barnard
authors.forename: Tillman U
authors.initials: TU
authors.lastname: Gerngross
mesh_terms: D043303:Aryldialkylphosphatase / Q000096:biosynthesis* / Q000235:genetics*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000502:physiology; D020118:Cupriavidus necator / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D013045:Species Specificity
chemical_list: D011994:Recombinant Proteins; D043303:Aryldialkylphosphatase