Increasing cell‐free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA‐binding proteins
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Sep 01, 2020
- Source ID
- 10.1002/bit.27538
Entities
People
- Bo Zhu
- Hideo Nakano
- Maria D Cabezas
- Michael C Jewett
- Robert Nicol
- Rui Gan
- Takaaki Kojima
Organizations
- Army Research Office
- Broad Institute
- David and Lucile Packard Foundation
- Defense Advanced Research Projects Agency
- Japan Society for the Promotion of Science
- Nagoya University
- National Institutes of Health
- Northwestern University