Abstract
Cell-free protein synthesis enables the rapid production and engineering of recombinant proteins. Existing cell-free systems differ substantially from each other with respect to efficiency, scalability and the ability to produce functional eukaryotic proteins. Here we describe species-independent translational sequences (SITS) that mediate efficient cell-free protein synthesis in multiple prokaryotic and eukaryotic systems, presumably through bypassing the early translation initiation factors. We use these leaders in combination with targeted suppression of the endogenous Leishmania tarentolae mRNAs to create a cell-free system based on this protozoan. The system can be directly programmed with unpurified PCR products, enabling rapid generation of large protein libraries and protein variants. L. tarentolae extract can produce up to 300 μg/ml of recombinant protein in 2 h. We further demonstrate that protein-protein and protein–small molecule interactions can be quantitatively analyzed directly in the translation mixtures using fluorescent (cross-) correlation spectroscopy.
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Acknowledgements
This work was supported in part by a Deutsche Forschungsgemeinschaft grant AL 484/5-4 and Heisenberg fellowship to K.A. U.T.T.N was supported by the predoctoral fellowship of Fonds der chemischen Industrie. We thank P. Lommerse for help with FCCS measurements and to V. Rajagopalan, M. Terbeck and A. Sander for excellent technical assistance. We are grateful to M. Gruen, R. Breitling, F. Seebeck and A. Goodall for critical reading of the manuscript and stimulating discussions and K. Browning (University of Texas at Austin) for a gift of reagents. We are very grateful to C. Clayton, S. Beverley, L. Shaloiko, I. Granowsky and P. Dittrich for support in the early stages of the project. We are indebted to R.S. Goody for long-term support.
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S.M. designed and performed research, O.K. designed and performed research, U.T.T.N designed and performed research and K.A. designed research and wrote the manuscript.
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Kirill Alexandrov is a stakeholder in Jena Bioscience, which markets Leishmania tarentolae-based in vivo protein expression systems.
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Mureev, S., Kovtun, O., Nguyen, U. et al. Species-independent translational leaders facilitate cell-free expression. Nat Biotechnol 27, 747–752 (2009). https://doi.org/10.1038/nbt.1556
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DOI: https://doi.org/10.1038/nbt.1556
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