T7Max transcription system.
cell-free protein expression
in vitro transcription
in vitro translation
synthetic cells
Journal
Journal of biological engineering
ISSN: 1754-1611
Titre abrégé: J Biol Eng
Pays: England
ID NLM: 101306640
Informations de publication
Date de publication:
23 Jan 2023
23 Jan 2023
Historique:
received:
17
10
2021
accepted:
04
01
2023
entrez:
23
1
2023
pubmed:
24
1
2023
medline:
24
1
2023
Statut:
epublish
Résumé
Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.
Sections du résumé
BACKGROUND
BACKGROUND
Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits.
RESULTS
RESULTS
Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates.
CONCLUSIONS
CONCLUSIONS
The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.
Identifiants
pubmed: 36691081
doi: 10.1186/s13036-023-00323-1
pii: 10.1186/s13036-023-00323-1
pmc: PMC9872363
doi:
Types de publication
Journal Article
Langues
eng
Pagination
4Subventions
Organisme : NIH HHS
ID : 5R01MH114031
Pays : United States
Organisme : NIMH NIH HHS
ID : R01 MH114031
Pays : United States
Informations de copyright
© 2023. The Author(s).
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