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The Skaggs Institute
for Chemical Biology
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.
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