Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification.
Alkaline lysis
Barnase
Cation-exchange chromatography
Plasmid
RNase
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
28 Aug 2021
28 Aug 2021
Historique:
received:
11
05
2021
accepted:
21
07
2021
entrez:
29
8
2021
pubmed:
30
8
2021
medline:
21
12
2021
Statut:
epublish
Résumé
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. 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 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.
Sections du résumé
BACKGROUND
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
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
CONCLUSIONS
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.
Identifiants
pubmed: 34454498
doi: 10.1186/s12934-021-01642-y
pii: 10.1186/s12934-021-01642-y
pmc: PMC8403359
doi:
Substances chimiques
Bacterial Proteins
0
barstar protein, Bacillus amyloliquefaciens
37328-61-3
Ribonucleases
EC 3.1.-
Bacillus amyloliquefaciens ribonuclease
EC 3.1.27.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
171Commentaires et corrections
Type : ErratumIn
Informations de copyright
© 2021. The Author(s).
Références
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