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          Institute: MPI für molekulare Genetik     Collection: Ribosome Research Groups     Display Documents

ID: 305644.0, MPI für molekulare Genetik / Ribosome Research Groups
Troubleshooting coupled in in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins
Authors:Iskakova, Madina B.; Szaflarski, Witold; Dreyfus, Marc; Remme, Jaanus; Nierhaus, Knud
Date of Publication (YYYY-MM-DD):2006-06-15
Title of Journal:Nucleic Acids Research
Journal Abbrev.:Nucl. Acids Res.
Issue / Number:19
Start Page:e135
End Page:e135
Copyright:© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Review Status:not specified
Audience:Experts Only
Abstract / Description:Cell-free coupled transcription–translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 ± 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20°C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20°C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.
Comment of the Author/Creator:To whom correspondence should be addressed. Tel: +49 30 8413 1700; Fax: +49 30 8413 1594; Email: nierhaus@molgen.mpg.de

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
External Publication Status:published
Document Type:Article
Version Comment:Automatic journal name synchronization
Communicated by:Ribosome Group
Affiliations:MPI für molekulare Genetik
External Affiliations:1.Laboratoire de Genetique Moleculaire, Centre National de la Recherche Scientifique UMR8541 Ecole Normale Superieure, 46 Rue d'Ulm, 75230 Paris Cedex 05, France;
2 Department of Molecular Biology, Institute of Molecular and Cell Biology, Tartu University Riia 23, 51010 Tartu, Estonia.
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