Increasing cell‐free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA‐binding proteins. Issue 12 (1st September 2020)

• Record Type: Journal Article
• Authors: Zhu, Bo; Gan, Rui; Cabezas, Maria D.; Kojima, Takaaki; Nicol, Robert; Jewett, Michael C.; Nakano, Hideo
• Is Part Of: Biotechnology and bioengineering. Volume 117:Issue 12(2020)
• Page Start: 3849
• Page End: 3857
• Publication Date: 2020-09-01
• Subjects: cell‐free protein synthesis -- CroP‐LET -- in vitro transcription/translation -- linear expression template -- scCro -- Vibrio natriegens; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27538
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: 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 in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. 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 the Vibrio natriegens CFPS platform. With the fast‐expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates. Abstract : In cell‐free protein synthesis (CFPS) reactions, linear expression templates (LET) offers unique applications and advantages over their circular counterparts; however, the low stability of the LETs in crude extract‐based reactions limited its usage. This work describes a generalizable method, named CroP‐LET, which protects the LETs with DNA‐binding proteins. The authors demonstrated that CroP‐LET increased the superfolder green fluorescent protein yields by 18‐fold and 6‐fold in the Vibrio natriegens and Escherichia coli CFPS platforms, respectively.