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

Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA.
Trends in Biotechnology (Impact Factor: 9.66). 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: In this report, we describe that the use of GroEL/GroES-enriched S30 extract remarkably enhances the solubility and enzymatic activity of cell-free synthesized rPA, which requires the correct formation of 9 disulfide bonds for its biological activity. We found that the stable maintenance of redox potential is necessary, but not sufficient for the optimal expression of active rPA. In a control reaction without using additional molecular chaperones, most of the rPA molecules were aggregated almost instantly after their expression and thus failed to exhibit the enzymatic activity. However, by the use of GroEL/GroES-enriched extract, combined with IAM-treatment, approximately of active rPA was expressed in the cell-free synthesis reaction. This result not only demonstrates the efficient production of complex proteins, but also shows the control and flexibility offered by the cell-free protein synthesis system.
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    ABSTRACT: Bacterial extracts are widely used to synthesize recombinant proteins. Vast data volumes have been accumulated in cell-free expression databases, covering a whole range of existing proteins. It makes possible comprehensive bioinformatics analysis and identification of multiple features associated with protein solubility and aggregation. In the present paper, an approach to identify the multiple physicochemical and structural properties of amino acid sequences associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts is presented. The method includes: (1) categorical assessment of expression data; (2) calculation and prediction of multiple properties of expressed sequences; (3) correlation of the individual properties with the expression scores; and (4) evaluation of statistical significance of the observed correlations. Using this method, a number of significant correlations between calculated and predicted properties of amino acid sequences and their propensity for soluble cell-free expression have been revealed.
    Frontiers in microbiology. 01/2014; 5:295.
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    ABSTRACT: Sophisticated cell-free protein synthesis (CFPS) systems have been developed as an alternative to recombinant expression in cultured cells. In this review, we present advances in the field of mammalian-based CFPS by highlighting recently established systems derived from mouse fibroblasts, HeLa, hybridoma, CHO and K562 cells. We further highlight ongoing challenges in the field of mammalian-based CFPS, such as the optimization of already established platforms and the development of novel systems in order to further increase protein yields and reduce manufacturing costs while facilitating the synthesis of a huge number of biologically active target proteins. Advances in mammalian-based CFPS shall expand the number of future applications of CFPS in the area of pharmaceutical research and development.
    Pharmaceutical Bioprocessing. 10/2014; Pharm. Bioprocess.((2014) 2(4)):339-348.


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