Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 27128597.
- Also identified by DOI 10.1371/journal.pone.0154614 and PMC identifier 4851396.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
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).
Medical subject headings
- Cell Extracts
- Cell-Free System
- Protein Biosynthesis