Increasing cell‐free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA‐binding proteins
| Autor: | Maria D. Cabezas, Bo Zhu, Robert Nicol, Michael C. Jewett, Takaaki Kojima, Hideo Nakano, Rui Gan |
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| Rok vydání: | 2020 |
| Předmět: |
Green Fluorescent Proteins
Bioengineering Vibrio natriegens medicine.disease_cause Applied Microbiology and Biotechnology DNA-binding protein chemistry.chemical_compound Plasmid Gene expression Escherichia coli medicine Polymerase Vibrio Cell-free protein synthesis Cell-Free System biology Escherichia coli Proteins biology.organism_classification DNA-Binding Proteins chemistry Biochemistry biology.protein DNA Plasmids Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 117:3849-3857 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.27538 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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