A novel knock out strategy to enhance recombinant protein expression in Escherichia coli.
Asparaginase
/ biosynthesis
Escherichia coli
/ genetics
Escherichia coli Proteins
/ genetics
Fermentation
Gene Expression Regulation, Bacterial
Gene Knockout Techniques
/ methods
Genes, Bacterial
Green Fluorescent Proteins
/ biosynthesis
Metabolic Networks and Pathways
/ genetics
Recombinant Proteins
/ biosynthesis
Signal Transduction
/ genetics
Stress, Physiological
Up-Regulation
Cellular stress response
Escherichia coli
Knock out
Recombinant protein expression
Signalling control
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
23 Jul 2020
23 Jul 2020
Historique:
received:
17
05
2020
accepted:
13
07
2020
entrez:
25
7
2020
pubmed:
25
7
2020
medline:
30
4
2021
Statut:
epublish
Résumé
The expression of recombinant proteins triggers a stress response which downregulates key metabolic pathway genes leading to a decline in cellular health and feedback inhibition of both growth and protein expression. Instead of individually upregulating these downregulated genes or improving transcription rates by better vector design, an innovative strategy would be to block this stress response thereby ensuring a sustained level of protein expression. We postulated that the genes which are commonly up-regulated post induction may play the role of signalling messengers in mounting the cellular stress response. We identified those genes which have no known downstream regulatees and created knock outs which were then tested for GFP expression. Many of these knock outs showed significantly higher expression levels which was also sustained for longer periods. The highest product yield (Y Creating key knock outs to block the CSR and enhance expression is a radically different strategy that can be synergistically combined with traditional methods of improving protein yields thus helping in the design of superior host platforms for protein expression.
Sections du résumé
BACKGROUND
BACKGROUND
The expression of recombinant proteins triggers a stress response which downregulates key metabolic pathway genes leading to a decline in cellular health and feedback inhibition of both growth and protein expression. Instead of individually upregulating these downregulated genes or improving transcription rates by better vector design, an innovative strategy would be to block this stress response thereby ensuring a sustained level of protein expression.
RESULTS
RESULTS
We postulated that the genes which are commonly up-regulated post induction may play the role of signalling messengers in mounting the cellular stress response. We identified those genes which have no known downstream regulatees and created knock outs which were then tested for GFP expression. Many of these knock outs showed significantly higher expression levels which was also sustained for longer periods. The highest product yield (Y
CONCLUSION
CONCLUSIONS
Creating key knock outs to block the CSR and enhance expression is a radically different strategy that can be synergistically combined with traditional methods of improving protein yields thus helping in the design of superior host platforms for protein expression.
Identifiants
pubmed: 32703203
doi: 10.1186/s12934-020-01407-z
pii: 10.1186/s12934-020-01407-z
pmc: PMC7376861
doi:
Substances chimiques
Escherichia coli Proteins
0
Recombinant Proteins
0
Green Fluorescent Proteins
147336-22-9
Asparaginase
EC 3.5.1.1
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
148Subventions
Organisme : NIDDK NIH HHS
ID : DK075081
Pays : United States
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