High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification.
We report further development of a novel recombinant protein expression system based on the Gram-negative bacterium, Ralstonia eutropha. In this study, we were able to express soluble, active, organophosphohydrolase (OPH), a protein that is prone to inclusion body formation in Escherichia coli, at titers greater than 10 g/L in high cell density fermentation. This represents a titer that is approximately 100-fold greater than titers previously reported in E. coli for this enzyme. R. eutropha strains expressing OPH were generated in two cloning steps. First, the T7 RNA polymerase gene was placed under the control of the strong, inducible phaP promoter and integrated into the phaP locus of R. eutropha NCIMB 40124. Second, a single copy of the oph gene under control of the T7 promoter was randomly integrated into the chromosome using a transposon cloning vector.
Barnard
Gavin C
GC
Thayer School of Engineering & Department of Biological Sciences, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755, USA.
Henderson
Grant E
GE
Srinivasan
Sriram
S
Gerngross
Tillman U
TU
D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000201:enzymology* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D010744:Phosphoric Monoester Hydrolases