Abstract
Cell-free protein synthesis (CFPS) is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20–30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL).
Highlights
Data Availability Statement: All relevant data are within the paper and its Supporting Information files
Crude cell extracts prepared by lysozyme treatment and freeze-thawing (LoFT cell extract)
These results suggest that LoFT cell extract requires more species of small metabolites doi:10.1371/journal.pone.0154614.g004
Summary
Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The most commonly used tools for the preparation of cell lysates for CFPS are the French press and bead crushers [26,27,31,32] This equipment is expensive, and not generally available in the majority of laboratories. Freeze-thaw cycles extract intracellular components by damaging the biological membranes These methods have been widely used to prepare cell extracts to analyze recombinant protein expression; a recent report has shown that cell extracts prepared by lysozyme treatment or freezethawing do not show efficient CFPS activity [28]. We report a method to prepare cell extract for CFPS using biochemical treatments This method employs a combination of lysozyme treatment and freeze-thawing, with highspeed centrifuges being the only required machinery. Because it is straightforward and scalable, our biochemical disruption method could provide an alternative to physical disruption methods developed so far
Sign-in/Register to view highlights & summary 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.
