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Species-independent translational leaders facilitate cell-free expression

Nature Biotechnology volume 27, pages 747–752 (2009)Cite this article

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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Figure 1: Design of unstructured species-independent translational sequences (SITS) and analysis of their ability to initiate translation in cell-free expression systems.
Figure 2: Preparation of cell-free translational lysate based on L. tarentolae.
Figure 3: Development of coupled LTE system.
Figure 4: Fluorescence correlation (FCS) and cross-correlation (FCCS) analysis of fluorescent proteins synthesized using the LTE system.

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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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Authors and Affiliations

  1. Institute for Molecular Bioscience and Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia

    Sergei Mureev, Oleksiy Kovtun & Kirill Alexandrov

  2. Max-Planck-Institute for Molecular Physiology, Dortmund, Germany

    Uyen T T Nguyen & Kirill Alexandrov

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  1. Sergei Mureev
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  2. Oleksiy Kovtun
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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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Correspondence to Kirill Alexandrov.

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Competing interests

Kirill Alexandrov is a stakeholder in Jena Bioscience, which markets Leishmania tarentolae-based in vivo protein expression systems.

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Supplementary Figures 1–16 and Supplementary Methods (PDF 1655 kb)

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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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