The past, present and future of cell-free protein synthesis.

Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA.
Trends in Biotechnology (Impact Factor: 10.04). 04/2005; 23(3):150-6. DOI: 10.1016/j.tibtech.2005.01.003
Source: PubMed

ABSTRACT Recent technical advances have revitalized cell-free expression systems to meet the increasing demands for protein synthesis. Cell-free systems offer several advantages over traditional cell-based expression methods, including the easy modification of reaction conditions to favor protein folding, decreased sensitivity to product toxicity and suitability for high-throughput strategies because of reduced reaction volumes and process time. Moreover, improvements in translation efficiency have resulted in yields that exceed a milligram of protein per milliliter of reaction mix. We review the advances on this expanding technology and highlight the growing list of associated applications.

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    ABSTRACT: 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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    ABSTRACT: Current, cell‑free methodologies allow for robust and rapid protein production and screening. The use of protein arrays attached selectively or nonspecifically to various solid supports is rapidly becoming a common research tool to explore the function and potential relationships of proteins encoded within any genome. Array‑based approaches are also ideal for parallel analysis of multiple binary interactions between proteins and other molecules. In addition, engineering novel tagging techniques allows the orientation of proteins of interest and expands the capabilities and use of protein microarrays. Combining cell‑free expression with array‑based proteomics promises to be a powerful tool for protein biochemistry, molecular diagnostics and therapeutics.
    Cell-Free Protein Expression, 1st Edition edited by W. Antoni Kudlicki, 01/2007: chapter Chapter12: pages 145-154; Landes Biosciences., ISBN: 978-1-58706-123-3
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    ABSTRACT: Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyse the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximise throughput and minimise the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in L. tarentolae and E. coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014. Published by Elsevier B.V.
    Journal of Biotechnology 12/2014; DOI:10.1016/j.jbiotec.2014.12.006 · 2.88 Impact Factor


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