Abstract
Cell-free protein synthesis (CFPS) has the advantage of rapid expression of proteins and has been widely implemented in synthetic biology and protein engineering. However, the critical problem limiting CFPS industrial application is its relatively high cost, which partly attributes to the overexpense of single-use DNA templates. Hydrogels provide a possible solution because they can preserve and reutilize the DNA templates in CFPS and have great potential in elevating the protein production yield of the CFPS. Here, we presented a low-cost hybrid hydrogel simply prepared with polyethylene glycol diacrylate (PEGDA) and DNA, which is capable of high-efficient and repeated protein synthesis in CFPS. Parameters governing protein production specific to hybrid hydrogels were optimized. Structures and physical properties of the hybrid hydrogel were characterized. Transcription and expression kinetics of solution phase system and gel phased systems were investigated. The results showed that PEGDA/DNA hydrogel can enhance the protein expression of the CFPS system and enable a repeated protein production for tens of times. This PEGDA/DNA hybrid hydrogel can serve as a recyclable gene carrier for either batch or continuous protein expression, and paves a path toward more powerful, scalable protein production and cell-free synthetic biology.
Highlights
Cell-free protein synthesis (CFPS) is an in vitro life simulation system that synthesizes proteins using cell extracted machinery, exogenous substrates, and DNA or RNA templates without the constraint of cells
The cross-linked polyethylene glycol diacrylate (PEGDA)/DNA hybrid hydrogel containing a certain amount of PEGDA and linear DNA was used for cell-free protein production in a solution containing 6.67 μl of cell-free lysate, 10.73 μl of the reaction buffer, and nucleasefree water
To prepare the hybrid hydrogel DNA component, which contained the gene template for protein expression, a pair of 5′-acrydite modified oligonucleotide primers was used to PCR amplify linear DNA from a plasmid (Figure S1)
Summary
Cell-free protein synthesis (CFPS) is an in vitro life simulation system that synthesizes proteins using cell extracted machinery, exogenous substrates, and DNA or RNA templates without the constraint of cells. (Caschera and Noireaux, 2014; Fujiwara and Doi, 2016; Schoborg et al, 2016) These efforts, improved the protein production of CFPS to a certain degree. Vast DNA consumption in large-scale reactions is still one of the bottlenecks in limiting the development of CFPS industrialization. To further reduce the DNA consumption in protein producing hydrogel, in this study, we successfully constructed a PEGDA/DNA hybrid hydrogel for CFPS. The cost of PEGDA/DNA hybrid hydrogel was reduced more than 30 times compared to that of Pgel. The optimized PEGDA/DNA hybrid hydrogel enhanced protein production 22.7-fold over the solution phased reactions. It was proved that the PEGDA/DNA hybrid hydrogel can be reused 10 times in CFPS, which showed great potential in large-scale CFPS application
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
