A simple and rapid method for preparing a cell-free bacterial lysate for protein synthesis

Nitzan Krinsky, Maya Kaduri, Janna Shainsky-Roitman, Mor Goldfeder, Eran Ivanir, Itai Benhar, Yuval Shoham, Avi Schroeder

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45 °C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

Original languageEnglish
Article numbere0165137
JournalPLoS ONE
Volume11
Issue number10
DOIs
StatePublished - Oct 2016

Funding

FundersFunder number
Seventh Framework Programme680242, 333797
Seventh Framework Programme

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