Author:
Deich Christopher,Cash Brock,Sato Wakana,Sharon Judee,Aufdembrink Lauren,Gaut Nathaniel J.,Heili Joseph,Stokes Kaitlin,Engelhart Aaron E.,Adamala Katarzyna P.
Abstract
Abstract
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
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
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.
Funder
National Science Foundation
John Templeton Foundation
NASA Headquarters
National Institutes of Health
Publisher
Springer Science and Business Media LLC
Subject
Cell Biology,Molecular Biology,Biomedical Engineering,Environmental Engineering
Cited by
8 articles.
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