Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use.
The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.
Voss
Carsten
C
Chair of Fermentation Engineering, Faculty of Technology, Bielefeld University, PO 100 131, D-33501 Bielefeld, Germany. cvo@fermtech.techfak.uni-bielefeld.de
Lindau
Dennis
D
Flaschel
Erwin
E
D000818:Animals; D001426:Bacterial Proteins; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005822:Genetic Vectors / Q000096:biosynthesis; D004364:Pharmaceutical Preparations; D010957:Plasmids / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology; D012260:Ribonucleases / Q000096:biosynthesis* / Q000302:isolation & purification
D001426:Bacterial Proteins; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D004247:DNA; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D012259:Ribonuclease, Pancreatic