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The Skaggs Institute
for Chemical Biology


Scientific Report 2008




Engineering Eukaryotic Algal Chloroplasts for Production of Human Therapeutic Proteins and of Biofuels

S.P. Mayfield, M. Tran, A. Manuell, J. Marin-Navarro, M. Muto, P. Pettersson, P. Lee, B. Rasala, M. Jager

Algae offer a tremendous opportunity as a biotechnology platform both for the production of protein therapeutics and as a source of renewable energy. Because production of therapeutic proteins in algae can be achieved at a fraction of the cost of traditional mammalian cell culture, algal production has the potential to dramatically reduce the cost of protein-based drugs. Algae can also produce biomass at 10 times the rate of terrestrial plants and can be grown in minimal media on land not suitable for food crop production, making algae a potential source of renewable biofuels.

To realize the potential of algae for production of biofuels and therapeutic proteins, we must understand and control algal gene expression. In algae, both proteins and biofuel molecules are produced in chloroplasts, and understanding chloroplast gene expression will be essential to developing algae as a biotechnology platform. Using molecular, biochemical, genetic, and structural biology, we have identified key factors that control gene expression within the chloroplast and have used this knowledge to produce algae that have strong, regulated protein expression. With this basic understanding of the genetics and biology of algae, we can develop eukaryotic algae as a cost-effective system for biotechnology applications, including the production of human therapeutic proteins, industrial enzymes, and biofuels.

Human Therapeutic Proteins

During the past several years, we have developed a system for the expression of recombinant proteins in the green alga Chlamydomonas reinhardtii. We now routinely obtain strong expression of complex mammalian proteins that are suitable as human therapeutic agents. We have expressed a number of proteins in algae, including monoclonal antibodies, growth factors, and a variety of other potential therapeutics.
In addition to therapeutic proteins, we have also expressed eukaryotic protein toxins, an achievement that is possible because chloroplasts are naturally resistant to such toxins. We have now developed antibody-toxin fusion proteins, a class of recombinant protein molecules that can target and kill eukaryotic cells, including human cancer cells. The production of antibody-toxin fusion proteins is unique to our expression system; bacterial expression systems cannot efficiently produce these complex molecules, and mammalian cell cultures would be killed by the toxin during production. Thus, algal chloroplasts are the best system for the production of this type of superior cancer therapeutic agent.

To examine the potential of algae to produce antibody-toxin fusion proteins as cancer therapeutics, we engineered an antibody to CD19 to fuse with an exotoxin-A protein domain to produce the antibody-toxin fusion protein anti-CD19–ETA, which targets human B-cell lymphomas. This recombinant fusion protein was expressed in algae, where it accumulated as a soluble protein. Using cell-based assays, we showed that isolated anti-CD19–ETA efficiently binds to CD19+ human B-cell lymphomas but does not bind to CD19 normal human cells. Once bound to the tumor cells, anti-CD19–ETA efficiently kills the cells. These cell-based assays are the first step in demonstrating the potential of these fusion proteins as human anticancer therapeutic agents.

Biofuels

With fossil fuel reserves dwindling, mandates requiring the reduction of carbon dioxide emissions, and a need for national energy independence, we face the formidable challenge of developing sustainable forms of carbon-neutral energy in an economically practical manner. Algae offer the potential to produce carbon-neutral liquid biofuels at a scale and cost that can be competitive with existing fossil fuel production. In addition, production of biofuels from algae will not compete with production of food crops for use of arable land. Economic production of biofuels from algae will require production of other molecules in addition to the biofuel molecules, because all of the biomass produced from algae will need to have a commercial value. The technology we developed for the production of therapeutic proteins in algae can be used to produce algae with valuable protein coproducts as well as improved biofuel characteristics, and we are now developing algae as a renewable energy source.

Publications

Beligni, M.V., Mayfield, S.P. Arabidopsis thaliana mutants reveal a role for CSP41a and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal RNA metabolism. Plant Mol. Biol. 67:389, 2008.

J.H., Siefker, D.T., Tran, M., Weber, A., McDonald, T.L., Mayfield, S.P. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant Biotechnol. J. 5:402, 2007.

 

Stephen P. Mayfield, Ph.D.
Professor
Associate Dean, Kellogg School of Science and Technology

Mayfield Web Site