Strategies to facilitate transgene expression in Chlamydomonas reinhardtii

  • Eichler-Stahlberg A
  • Weisheit W
  • Ruecker O
 et al. 
  • 147

    Readers

    Mendeley users who have this article in their library.
  • 67

    Citations

    Citations of this article.

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.

Author-supplied keywords

  • Chlamydomonas reinhardtii
  • Erythropoietin
  • Gene expression
  • Intron
  • Luciferase
  • Secretion

Get free article suggestions today

Mendeley saves you time finding and organizing research

Sign up here
Already have an account ?Sign in

Find this document

Authors

  • Alke Eichler-Stahlberg

  • Wolfram Weisheit

  • Ovidiu Ruecker

  • Markus Heitzer

Cite this document

Choose a citation style from the tabs below

Save time finding and organizing research with Mendeley

Sign up for free