Article

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

Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA.
Trends in Biotechnology (Impact Factor: 11.96). 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.

    • "Cell-free protein synthesis (CFPS) systems have emerged as a powerful technology platform for rapid and efficient production of medicinal proteins [4] [5] [6] [7] [8]. Such systems have distinct advantages over in vivo methods for recombinant protein production [9] [10] [11] [12]. Cell-free systems do not require ancillary processes for cell viability and growth, allowing optimization of manufacture for a single protein product. "
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    ABSTRACT: Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.
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    • "Other problems arising from the necessity of a vital cellular host are directed targeting and translocation of membrane proteins to their final membrane destination, proteolytic attack by protein control systems and overloading of protein transport mechanisms (Schwarz et al., 2007). Lacking the boundaries of a cellular plasma membrane and being fast, scalable and easy to handle, CFPS can be considered a promising alternative to classical cell-based membrane protein production (Katzen et al., 2005). The applicability of cell-free membrane protein synthesis based on prokaryotic extracts has been demonstrated in various examples. "

    Full-text · Dataset · Apr 2015
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    • "Other problems arising from the necessity of a vital cellular host are directed targeting and translocation of membrane proteins to their final membrane destination, proteolytic attack by protein control systems and overloading of protein transport mechanisms (Schwarz et al., 2007). Lacking the boundaries of a cellular plasma membrane and being fast, scalable and easy to handle, CFPS can be considered a promising alternative to classical cell-based membrane protein production (Katzen et al., 2005). The applicability of cell-free membrane protein synthesis based on prokaryotic extracts has been demonstrated in various examples. "
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    ABSTRACT: Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents a valuable alternative that allows for the high-throughput synthesis of functional membrane proteins. Here, we demonstrate the potential of our cell-free protein synthesis system, based on lysates from cultured Spodoptera frugiperda 21 cells, to produce pro- and eukaryotic membrane proteins with individual topological characteristics in an automated fashion. Analytical techniques, including confocal laser scanning microscopy, fluorescence detection of eYFP fusion proteins in a microplate reader and in-gel fluorescence of statistically incorporated fluorescent amino acid derivatives were employed. The reproducibility of our automated synthesis approach is underlined by coefficients of variation below 7.2 %. Moreover, the functionality of the cell-free synthesized potassium channel KcsA was analyzed electrophysiologically. Finally, we expanded our cell-free membrane protein synthesis system by an orthogonal tRNA/synthetase pair for the site-directed incorporation of p-Azido-L-phenylalanine based on stop codon suppression. Incorporation was optimized by performance of a two-dimensional screening with different Mg(2+) and lysate concentrations. Subsequently, the selective modification of membrane proteins with incorporated p-Azido-L-phenylalanine was exemplified by Staudinger ligation with a phosphine-based fluorescence dye. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Mar 2015 · Journal of Biotechnology
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