화학공학소재연구정보센터
Biotechnology and Bioengineering, Vol.117, No.12, 3849-3857, 2020
Increasing cell-free gene expression yields from linear templates inEscherichia coliandVibrio natriegensextracts by using DNA-binding proteins
In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs inEscherichia coliCFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization inE. coliCFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in theVibrio natriegensCFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.