title: Strategies to facilitate transgene expression in Chlamydomonas reinhardtii.
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.
authors.affiliation: Center of Excellence for Fluorescent Bioanalysis, University of Regensburg, Josef-Engert-Str. 9, 93053, Regensburg, Germany.
authors.forename: Alke
authors.initials: A
authors.lastname: Eichler-Stahlberg
authors.forename: Wolfram
authors.initials: W
authors.lastname: Weisheit
authors.forename: Ovidiu
authors.initials: O
authors.lastname: Ruecker
authors.forename: Markus
authors.initials: M
authors.lastname: Heitzer
mesh_terms: D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D015870:Gene Expression; D015151:Immunoblotting; D007438:Introns / Q000235:genetics; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D049410:Luciferases, Renilla; D012273:Ribulose-Bisphosphate Carboxylase