News and Publications

Regulation of Translation and Expression of Human Monoclonal Antibodies in Chloroplasts

In plants and algae, expression of many genes is regulated in response to exposure to light. Biochemical and genetic analyses revealed that light activates translation of plant mRNAs and that both RNA elements and their cognate RNA-binding proteins are required for this translational regulation. We identified several RNA elements required for chloroplast translation, including a ribosome-binding sequence (RBS) similar to sequences found in bacteria. Chloroplast RBS elements are required for association of mRNAs that encode thylakoid membrane proteins but not for translation of mRNAs that encode soluble proteins. In addition, the RBS of chloroplast mRNAs is positioned farther upstream than is the RBS of bacterial mRNAs, suggesting a fundamental difference between chloroplasts and bacteria in the mechanism by which an RBS facilitates initiation of translation. These data suggest that 2 independent mechanisms exist in chloroplasts for regulating mRNA translation. We are examining the chloroplast ribosome and ribosome-associated proteins to identify regulatory components involved in these processes.

Using a proteomic approach, we identified the complete set of chloroplast ribosomal proteins from Chlamydomonas reinhardtii. We also identified a number of ribosome-associated proteins and mRNA-specific RNA-binding proteins. The set of mRNA- binding proteins includes proteins homologous to poly(A)-binding proteins and protein disulfide isomerases, which together can modulate mRNA-binding activity in a light-dependent manner. On the basis of these studies, we propose the following model for translational activation: Redox potential, generated by the light reactions of photosynthesis, is used by chloroplast protein disulfide isomerase to activate binding of the chloroplast poly(A)-binding protein, and other RNA-binding proteins, to the 5´ untranslated region of chloroplast mRNAs. Binding of these proteins to RNA elements allows increased ribosome association and initiation of translation.

We are using proteomic and genetic analysis to identify the complete set of proteins and RNA elements required for chloroplast translation. Structural analysis of these RNA-protein complexes is being used to determine how RNA-protein interactions enhance ribosome binding and membrane association as a means to regulate translational activation.

Using our understanding of chloroplast translation, we developed a system for expressing a wide array of recombinant proteins, including those used as human therapeutic agents. For example, we constructed a strain of C reinhardtii that expresses a large single-chain antibody directed against herpes simplex virus. This antibody assembles in the cell to form a fully functional antibody that binds herpes simplex proteins. Antibody binding to the coat protein of herpes simplex virus can block viral propagation. We also showed that this antibody assembles into dimers, a key step for the proper function of many antibody molecules. In addition, we developed 2 reporter gene systems, 1 based on green fluorescent protein and 1 on bacterial luciferase, that will allow us to better define the transcriptional and translational elements required to obtain high levels of antibody expression. These studies indicate that eukaryotic algae have tremendous potential for the expression of recombinant human therapeutic proteins, because large-scale growth of algae is economically feasible.

Publications

Barnes, D., Mayfield, S.P. Redox control of posttranscriptional processes in the chloroplast. Antioxid. Redox Signal. 5:89, 2003.

Franklin, S., Ngo, B., Efuet, E., Mayfield, S.P. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J. 30:733, 2002.

Kim, J., Mayfield, S.P. The active site of the thioredoxin-like domain of chloroplast protein disulfide isomerase, RB60, catalyzes the redox-regulated binding of chloroplast poly(A)-binding protein, RB47, to the 5´ untranslated region of psbA mRNA. Plant Cell Physiol. 43:1238, 2002.

Mayfield, S.P., Franklin, S.E., Lerner, R.A. Expression and assembly of a fully active antibody in algae. Proc. Natl. Acad. Sci. U. S. A. 100:438, 2003.

Yamaguchi, K., Mayfield, S.P., Sugita, M. Transcriptional and translational regulation in biosynthesis and repair. In: Photosystem II. Wydrzyaski, T., Satoh, K. (Eds.). Kluwer, New York, in press.

Yamaguchi, K., Prieto, S., Beligni, M.V., Haynes, P.A., McDonald, W.H., Yates, J.R. III, Mayfield, S.P. Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome: identification of a novel S1 domain-containing protein and unusually large orthologs of bacterial S2, S3, and S5. Plant Cell. 14:2957, 2002.