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Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

1
Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Trubetskaya str. 8, bld. 2, 119991 Moscow, Russia
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(3), 928; https://doi.org/10.3390/ijms21030928
Received: 29 November 2019 / Revised: 15 January 2020 / Accepted: 28 January 2020 / Published: 31 January 2020
Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production. View Full-Text
Keywords: recombinant expression; cell-free (CF) protein synthesis; membrane proteins; disulfide bond; protein folding; noncanonical amino acids (NAAs); post-translational modification (PTM) recombinant expression; cell-free (CF) protein synthesis; membrane proteins; disulfide bond; protein folding; noncanonical amino acids (NAAs); post-translational modification (PTM)
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Smolskaya, S.; Logashina, Y.A.; Andreev, Y.A. Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis. Int. J. Mol. Sci. 2020, 21, 928.

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