Abstract
Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/ mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.
Original language | English (US) |
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Article number | e58882 |
Journal | Journal of Visualized Experiments |
Volume | 2019 |
Issue number | 144 |
DOIs | |
State | Published - Feb 2019 |
Funding
Authors would like to acknowledge Dr. Jennifer VanderKelen, Andrea Laubscher, and Tony Turretto for technical support, Wesley Kao, Layne Williams, and Christopher Hight for helpful discussions. Authors also acknowledge funding support from the Bill and Linda Frost Fund, Center for Applications in Biotechnology's Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF-1708919). MZL acknowledges the California State University Graduate Grant. MCJ acknowledges the Army Research Office W911NF-16-1-0372, National Science Foundation grants MCB-1413563 and MCB-1716766, the Air Force Research Laboratory Center of Excellence Grant FA8650-15-2-5518, the Defense Threat Reduction Agency Grant HDTRA1-15-10052/P00001, the David and Lucile Packard Foundation, the Camille Dreyfus Teacher-Scholar Program, the Department of Energy BER Grant DE-SC0018249, the Human Frontiers Science Program (RGP0015/2017), the DOE Joint Genome Institute ETOP Grant, and the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust for support.
Keywords
- CFME
- CFPS
- Cell-free metabolic engineering
- Cell-free protein synthesis
- Chemistry
- High-throughput protein synthesis
- Issue 144
- Synthetic biology
- TX-TL
- in vitro protein expression
ASJC Scopus subject areas
- General Chemical Engineering
- General Biochemistry, Genetics and Molecular Biology
- General Immunology and Microbiology
- General Neuroscience