Prospects for molecular farming in the green alga Chlamydomonas.

Protein-based therapeutics have enjoyed great success over the past decade. Unfortunately, this clinical success has come with a heavy price tag that is due to the inherently high costs of the capitalization and production of these complex molecules using current mammalian-based fermentation systems. Recent progress has been made in the production of recombinant proteins, including antibodies, in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. C. reinhardtii offers an attractive alternative to traditional mammalian-based expression systems for several reasons, including its ability to provide stable plastid and nuclear transformants rapidly and its inherently low costs for capitalization and production.

Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast.

In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.

Expression of human antibodies in eukaryotic micro-algae.

Protein based therapeutics have enjoyed great success over the past decade. Unfortunately, with this clinical success comes a heavy price tag, owing to the inherently high costs of capitalization and production using mammalian cell fermentation. To address this problem, we have begun developing a system for the expression of recombinant proteins in the unicellular eukaryotic green algae, Chlamydomonas reinhardtii, leading to the production of human IgA single chain antibodies. The expression of human monoclonal antibodies in C. reinhardtii offers an attractive alternative to traditional mammalian based expression systems for several reasons, including an ability to rapidly obtain stable plastid and nuclear transformants, coupled with inherently low costs of capitalization and production.

Recent developments in the production of human therapeutic proteins in eukaryotic algae.

Antibody-based therapeutics have had great success over the last few years, and continue to be one of the fastest growing sectors of drug development. The efficacy and specificity of antibody-based drugs makes them ideal candidates for new drug development, but the specificity of these drugs comes from their complexity, and this complexity makes antibodies very expensive to produce. To address this problem, the authors have developed a system for the expression of recombinant proteins using the unicellular eukaryotic green algae, Chlamydomonas reinhardtii. As proof of concept, the authors have engineered microalgae to produce several forms of a human IgA antibody directed against herpes simplex virus. The expression of human monoclonal antibodies in C. reinhardtii offers an attractive alternative to traditional mammalian-based expression systems, as both the plastid and nuclear genomes are easily and quickly transformed, and the production of proteins in algae has an inherently low cost of capitalisation and production.

Expression and assembly of a fully active antibody in algae.

Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.