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The Skaggs Institute For Chemical Biology
Scientific Report 1999-2000


Expression of Antibodies in Microalgae


S.P. Mayfield, M. Gonzalez, D. Girard, E. Efuet, C. Fong, H.-C. Hung, B. Ngo, A. Somanchi, S.E. Franklin

The focus of our work is the development of a new system for expression of recombinant proteins that uses the eukaryotic green alga Chlamydomonas reinhardtii. Currently, a number of heterologous protein expression systems are available for the production of human therapeutic and value-added proteins. Each system has distinct advantages in terms of protein yield, ease of manipulation, and cost of operation.

The production of heterologous proteins in terrestrial plants (e.g., corn, soybeans) has received considerable attention in the past several years as the demand to economically produce valuable biopharmaceuticals on an extremely large scale has emerged. Such technology may make available therapies that were previously ignored simply because they were prohibitively expensive. Proteins produced in plant systems are also generally regarded as safe, posing little risk of contamination by viruses, prions, or bacterial endotoxins. In addition, higher plant systems have been used to produce protein complexes such as dimeric secretory immunoglobulin A molecules, a procedure that required the simultaneous expression and assembly of 4 separate gene products to generate the fully functional antibody. No other expression system (bacterial, yeast, or eukaryotic cell culture) is currently available for producing these types of complex molecules. Thus, plants are an attractive system for expression of recombinant proteins and are perhaps the only economic alternative for the expression of multimeric proteins such as antibodies.

However, the production of recombinant proteins in higher plants has drawbacks, which can make this system less attractive from an economic standpoint. The first drawback is the substantial length of time required from the initial transformation event to small-scale evaluation and production, often 2 years or more. This length is primarily due to the relatively slow growth rates of terrestrial plants relative to other organisms used for protein expression. Hence, the initial generation of transformants; their propagation to flowering, genetic crosses; and production of seed stocks are handicapped by this intrinsically slow rate of growth. Higher plant expression systems also are associated with complex processing issues, because recombinant proteins are produced and deposited in specific organs such as leaves, fruits, and seeds. These proteins must be purified to homogeneity out of a complex mixture of tissues and cell types, a requirement that can add significantly to the costs of purification.

Microalgae such as C reinhardtii share many attributes with higher plants, including the ability to transform both the nuclear and chloroplast genomes and a low risk of contamination by viruses, prions, or endotoxin. Unlike higher plants, however, microalgae have relatively fast growth rates, and primary transformants can be generated in as little as 2 weeks. Furthermore, cultures can achieve high cell densities and be grown in volumes in excess of 500,000 L. Finally, because microalgae are unicellular, concerns about processing proteins out of complex tissues are obviated, and the possibility exists that proteins can be secreted directly into the culture medium or targeted for sequestration in the periplasmic space.

We engineered strains of C reinhardtii that express antibody genes in both the chloroplast and the cytoplasm. We expressed 3 single-chain antibodies in the chloroplast: an antibody that recognizes epitopes of herpes simplex virus, another that recognizes tetanus toxin, and a catalytic aldolase antibody. Although these antibodies accumulate within the chloroplast, the levels are still fairly low. Our current efforts are directed at increasing the levels of expression of these constructs and at expressing full-length versions of these antibodies.

We also developed expression vectors for the synthesis and export of antibodies from the cell. Such vectors should direct the accumulation of functional antibodies in the culture medium, greatly facilitating purification of the antibodies. One such construct expresses a full-length antibody that recognizes herpes simplex virus and blocks infection by the virus. We are using a variety of transcriptional promoters and translational enhancers to increase the levels of expression from these constructs.

Our work to date indicates that microalgae can indeed be used to produce fully functional antibodies. We think that ultimately these organisms can be used to produce antibodies in a cost-effective manner, on a very large scale, and in a form that is easily purified and free of contamination by viruses, prions, or endotoxin.

Publications

Fong, C., Lentz, A., Mayfield, S.P. Disulfide bond formation between RNA binding domains is used to regulate mRNA binding activity of the chloroplast poly(A) binding protein, RB47. J. Biol. Chem. 275:8274, 2000.

Somanchi, A., Mayfield, S.P. Nuclear chloroplast signaling. Curr. Opin. Plant Biol. 2:404, 1999.

Somanchi, A., Mayfield, S.P. Regulation of chloroplast translation. In: Regulatory Aspects of Photosynthesis. Aro, E.-M., Anderson, B. (Eds.). Kluwer Academic, Boston, in press.

 

 







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