Shankar, Ram; Schäffer, Nina; Schmeer, Marco; Friehs, Karl; Risse, Joe Max ; Schleef, Martin: Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification. In: Microbial Cell Factories. Jg.20. 2021
Inhalt
- Barnase expression construct
- Batch fermentation
- Ammonium sulphate precipitation
- Cation exchange chromatography
- Hydrophobic interaction chromatography (HIC)
- HIC as a secondary purification step
- Removal of residual plasmid DNA
- Application
- RNase PF in plasmid production using anion-exchange chromatography
- Discussion
- Conclusions
- Methods
- Host Strain and plasmid
- Construction of plasmid PF1680
- Shake-flask cultivation
- Fermentation
- Downstream processing
- Cation exchange chromatography
- Scale up of SP Sepharose FF
- Hydrophobic interaction chromatography
- DNA removal
- Assay to test for RNase activity
- Enzyme activity quantification
- SDS PAGE
- Determination of total protein concentration
- Test of RNase activity in resuspended cells solution
- Application to DNA purification
- Acknowledgements
- References