title: A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.
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.
authors: Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion - Israel Institute of Technology, Haifa, Israel.|The Interdisciplinary Program for Biotechnology, Technion - Israel Institute of Technology, Haifa, Israel.
authors: Nitzan
authors: http://orcid.org/0000-0002-6104-255X
authors: N
authors: Krinsky
authors: Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion - Israel Institute of Technology, Haifa, Israel.
authors: Maya
authors: M
authors: Kaduri
authors: Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion - Israel Institute of Technology, Haifa, Israel.
authors: Janna
authors: J
authors: Shainsky-Roitman
authors: Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion - Israel Institute of Technology, Haifa, Israel.
authors: Mor
authors: M
authors: Goldfeder
authors: Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa, Israel.
authors: Eran
authors: E
authors: Ivanir
authors: Department of Molecular Microbiology and Biotechnology, The Georg S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.
authors: Itai
authors: I
authors: Benhar
authors: Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa, Israel.
authors: Yuval
authors: Y
authors: Shoham
authors: Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion - Israel Institute of Technology, Haifa, Israel.
authors: Avi
authors: A
authors: Schroeder
mesh_terms: D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis*
chemical_list: D029968:Escherichia coli Proteins