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Strategies to facilitate transgene expression in Chlamydomonas reinhardtii

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Abstract

The unicellular green alga Chlamydomonas reinhardtii has been identified as a promising organism for the production of recombinant proteins. While during the last years important improvements have been developed for the production of proteins within the chloroplast, the expression levels of transgenes from the nuclear genome were too low to be of biotechnological importance. In this study, we integrated endogenous intronic sequences into the expression cassette to enhance the expression of transgenes in the nucleus. The insertion of one or more copies of intron sequences from the Chlamydomonas RBCS2 gene resulted in increased expression levels of a Renilla-luciferase gene used as a reporter. Although any of the three RBCS2 introns alone had a positive effect on expression, their integration in their physiological number and order created an over-proportional stimulating effect observed in all transformants. The secretion of the luciferase protein into the medium was achieved by using the export sequence of the Chlamydomonas ARS2 gene in a cell wall deficient strain and Renilla-luciferase could be successfully concentrated with the help of attached C-terminal protein tags. Similarly, a codon adapted gene variant for human erythropoietin (crEpo) was expressed as a protein of commercial relevance. Extracellular erythropoietin produced in Chlamydomonas showed a molecular mass of 33 kDa probably resulting from post-translational modifications. Both, the increased expression levels of transgenes by integration of introns and the isolation of recombinant proteins from the culture medium are important steps towards an extended biotechnological use of this alga.

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Acknowledgments

We wish to thank Regina Groebner-Fererra for perfect technical assistance and Amparo Hausherr for the purification of luciferase from E. coli. This work has been financially supported in parts by the Bavarian Ministry of Economic Affairs, Infrastructure, Transport and Technology, the Bavarian Ministry of Environment, Public Health and Consumer Protection and the German Federal Ministry of Education and Research including an ExistSeed grant and a BioChance grant together with the Entelechon GmbH.

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  1. Markus Heitzer

    Present address: Geneart AG, Josef-Engert-Str. 11, 93053, Regensburg, Germany

Authors and Affiliations

  1. Center of Excellence for Fluorescent Bioanalysis, University of Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany

    Alke Eichler-Stahlberg, Wolfram Weisheit, Ovidiu Ruecker & Markus Heitzer

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  1. Alke Eichler-Stahlberg
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  2. Wolfram Weisheit
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Correspondence to Markus Heitzer.

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Eichler-Stahlberg, A., Weisheit, W., Ruecker, O. et al. Strategies to facilitate transgene expression in Chlamydomonas reinhardtii . Planta 229, 873–883 (2009). https://doi.org/10.1007/s00425-008-0879-x

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