Journal Article

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

Risse, Joe Max, Schleef, Martin, Shankar, Ram, Friehs, Karl, Schäffer, Nina, Schmeer, Marco

2021

Overview

Background The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL − 1 ), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Alkaline lysis / Analysis / Applied Microbiology / Bacterial Proteins - biosynthesis / Bacterial Proteins - metabolism / Barnase / Barstar / Biotechnology / Cation exchange / Cation exchanging / Cation-exchange chromatography / Chemical properties / Chemistry / Chemistry and Materials Science / Chromatography / Cloning / Cytoplasm / Deoxyribonucleic acid / DNA / E coli / Enzymes / Enzymology / Escherichia coli / Escherichia coli - metabolism / Experiments / Fermentation / Gene therapy / Genetic Engineering / Hydrophobicity / Inhibitors / Lysates / Microbial Genetics and Genomics / Microbiology / Periplasm / Plasmid / Plasmids / Product safety / Proteins / Purification / Recombinant microbial toxins / Reengineering / Ribonucleases - biosynthesis / RNase / Toxicity