Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption

PLoS One. 2016 Apr 29;11(4):e0154614. doi: 10.1371/journal.pone.0154614. eCollection 2016.

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotechnology / methods
  • Cell Extracts / isolation & purification*
  • Cell-Free System / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Freezing
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / isolation & purification
  • Muramidase
  • Osmotic Pressure
  • Protein Biosynthesis*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Synthetic Biology

Substances

  • Cell Extracts
  • Recombinant Proteins
  • Green Fluorescent Proteins
  • Muramidase

Grant support

This work was supported by JSPS KAKENHI grants (Grant Numbers 11J03718, 26650044, and 15H00826) https://www.jsps.go.jp/english/e-grants/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.