Strategies to facilitate transgene expression in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> |
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Authors: | Alke Eichler-Stahlberg Wolfram Weisheit Ovidiu Ruecker Markus Heitzer |
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Institution: | (1) Center of Excellence for Fluorescent Bioanalysis, University of Regensburg, Josef-Engert-Str. 9, 93053 Regensburg, Germany;(2) Present address: Geneart AG, Josef-Engert-Str. 11, 93053 Regensburg, Germany |
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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.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Chlamydomonas reinhardtii Erythropoietin Gene expression Intron luciferase Secretion |
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