A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

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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.

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Most cited references 29

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Cell-free protein synthesis: applications come of age.

Erik Carlson, Rui Gan, C Hodgman … (2012)

Cell-free protein synthesis has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications. Copyright © 2011 Elsevier Inc. All rights reserved.

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High-throughput preparation methods of crude extract for robust cell-free protein synthesis

Yong-Chan Kwon, Michael Jewett (2015)

Crude extract based cell-free protein synthesis (CFPS) has emerged as a powerful technology platform for high-throughput protein production and genetic part characterization. Unfortunately, robust preparation of highly active extracts generally requires specialized and costly equipment and can be labor and time intensive. Moreover, cell lysis procedures can be hard to standardize, leading to different extract performance across laboratories. These challenges limit new entrants to the field and new applications, such as comprehensive genome engineering programs to improve extract performance. To address these challenges, we developed a generalizable and easily accessible high-throughput crude extract preparation method for CFPS based on sonication. To validate our approach, we investigated two Escherichia coli strains: BL21 Star™ (DE3) and a K12 MG1655 variant, achieving similar productivity (defined as CFPS yield in g/L) by varying only a few parameters. In addition, we observed identical productivity of cell extracts generated from culture volumes spanning three orders of magnitude (10 mL culture tubes to 10 L fermentation). We anticipate that our rapid and robust extract preparation method will speed-up screening of genomically engineered strains for CFPS applications, make possible highly active extracts from non-model organisms, and promote a more general use of CFPS in synthetic biology and biotechnology.

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Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system.

Ki Kim, Cha Choi, Gil-Chun Park … (2006)

In this study, as a part of our efforts to improve the robustness and economical feasibility of cell-free protein synthesis, we developed a simple method of preparing the cell extracts used for catalyzing cell-free protein synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support cell-free protein synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.

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Author and article information

Contributors

Thomas Preiss: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 21 October 2016

Publication date Collection: 2016

Volume: 11

Issue: 10

Electronic Location Identifier: e0165137

Affiliations

[1 ]Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion – Israel Institute of Technology, Haifa, Israel

[2 ]The Interdisciplinary Program for Biotechnology, Technion – Israel Institute of Technology, Haifa, Israel

[3 ]Department of Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa, Israel

[4 ]Department of Molecular Microbiology and Biotechnology, The Georg S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

John Curtin School of Medical Research, AUSTRALIA

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceptualization: NK MK JS MG AS IB YS.
Formal analysis: NK MK JS MG AS.
Funding acquisition: AS.
Investigation: NK MK JS MG AS.
Methodology: NK JS MG AS EI IB YS.
Resources: AS IB YS.
Supervision: AS.
Validation: NK MK JS MG AS.
Visualization: NK AS.
Writing – original draft: NK JS MG AS IB YS.
Writing – review & editing: NK JS MG AS IB YS.

* E-mail: avids@ 123456technion.ac.il

Author information

Nitzan Krinsky http://orcid.org/0000-0002-6104-255X

Article

Publisher ID: PONE-D-16-30787

DOI: 10.1371/journal.pone.0165137

PMC ID: 5074584

PubMed ID: 27768741

SO-VID: 59161bbe-90e2-47ce-b0f4-f20bef369d0d

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 1 August 2016

Date accepted : 6 October 2016

Page count

Figures: 4, Tables: 3, Pages: 13