Nucleic Acids Research Advance Access published online on October 11, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl462
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Methods Online |
Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins
Max-Planck-Institut für Molekulare Genetik, AG Ribosomen Ihnestrasse 73, D-14195 Berlin, Germany 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
*To whom correspondence should be addressed. Tel: +49 30 8413 1700; Fax: +49 30 8413 1594; Email: nierhaus{at}molgen.mpg.de
Received January 30, 2006. Revised May 9, 2006. Accepted June 15, 2006.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors