Journal
PLOS ONE
Volume 11, Issue 10, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0165137
Keywords
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Funding
- EU-FP7 Marie Curie Program - Israel Science Foundation [333797, 1778/13]
- Israel Cancer Association [20150116]
- Israel Ministry of Economy [52752]
- Israel Ministry of Science, Technology and Space [311878]
- GIF [1-2328-1139. 10/20]
- ERC [2015-STG-680242]
- Mallat Family Foundation
- Russell Berrie Nanotechnology Institute (RBNI) at the Technion - Lorry Lokey Interdisciplinary Center for Life Sciences & Engineering at the Technion - Alon Fellowship from the Council For Higher Education
- Taub Foundation Fellowships
- Baroness Ariane de Rothschild Women Doctoral Program from the Rothschild Caesarea Foundation
- Interdisciplinary Program for Biotechnology at the Technion
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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 mu g-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 degrees 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.
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