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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3849-3857 |
| Number of pages | 9 |
| Journal | Biotechnology and Bioengineering |
| Volume | 117 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 2020 |
Funding
We thank graduate student Weston Kightlinger in the Jewett Lab for the preparation of the GamS protein. We also thank Ashty Karim for his input on the manuscript. This study was supported in part by a Grant\u2010in\u2010Aid (no. 19H02523) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) to Hideo Nakano; Michael C. Jewett acknowledges support from the DARPA 1000 Molecules Program HR0011\u201015\u2010C\u20100084, Army Research Office Grants W911NF\u201016\u20101\u20100372, W911NF\u201019\u20101\u20100298, and W911NF\u201018\u20101\u20100200; the National Institutes of Health Grant 1U19AI142780\u201001, the David and Lucile Packard Foundation, and the Camille Dreyfus Teacher\u2010Scholar Program. We thank graduate student Weston Kightlinger in the Jewett Lab for the preparation of the GamS protein. We also thank Ashty Karim for his input on the manuscript. This study was supported in part by a Grant-in-Aid (no. 19H02523) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) to Hideo Nakano; Michael C. Jewett acknowledges support from the DARPA 1000 Molecules Program HR0011-15-C-0084, Army Research Office Grants W911NF-16-1-0372, W911NF-19-1-0298, and W911NF-18-1-0200; the National Institutes of Health Grant 1U19AI142780-01, the David and Lucile Packard Foundation, and the Camille Dreyfus Teacher-Scholar Program.
Keywords
- CroP-LET
- Vibrio natriegens
- cell-free protein synthesis
- in vitro transcription/translation
- linear expression template
- scCro
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Applied Microbiology and Biotechnology