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The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998


Expression of Antibodies and Novel Enzymatic Functions in Algae


S. Mayfield, J. Kim, E. Efuet, R. Bruick, A. Lentz, P. Choi, J. Allen, C. Fong

We express antibodies in algae for several reasons. For example, because of the advent of bacteria resistant to antibiotics, alternative treatments are essential to maintain the current standards of health care. Treatment of bacterial infections with antibodies is quite effective, but the cost of producing antibodies makes such treatment prohibitively expensive. Production of antibodies in algae should reduce these costs significantly and make antibody therapy a practical alternative to treatment with antibiotics. In addition, antibodies are protein molecules that can bind other molecules, both complex and simple, with high specificity and affinity. This attribute has made antibodies ideal molecules for a number of biotechnologic uses, and this specific binding of antibodies has been exploited by research groups to engineer antibody catalysts, including catalysts that do not occur naturally.

Because antibodies are increasingly used as therapeutic and research tools, the need to produce these proteins in pure form and in large quantities is obvious. Plants or algae can be used to produce these pharmacologically important proteins and enzymes on a large scale and in relatively pure form. Expression of antibody catalysts in algae will enable us to introduce novel enzymatic functions into these organisms to alter metabolites to produce new compounds.

Microalgae have several unique characteristics that make them ideal organisms for the production of antibodies. First, unlike most organisms and cells currently used to produce transgenic proteins, algae can be grown on a large scale in minimal media (inorganic salts) with sunlight as the energy source. Second, plants and algae have 2 distinct compartments, the cytoplasm and the chloroplast, in which proteins can be expressed. The cytoplasm of algae is similar to that of other eukaryotic organisms used for protein expression, such as yeast and insect cells. Chloroplasts are unique to plants and algae, and proteins expressed in this environment most likely will have properties different from those of cytoplasmically expressed proteins.

We engineered strains of the green alga Chlamydomonas reinhardtii to express antibody genes in the chloroplast. We expressed a truncated version of an antibody, known as single-chain antibody, that recognizes the tetanus toxin and a second antibody that is a catalytic aldolase. We also constructed a vector that contains a larger antibody composed of both a light-chain protein and a heavy-chain protein. Both the single-chain antibody and the larger antibody are capable of binding tetanus toxin, and in general these transgenically expressed antibodies have properties that appear identical to those of antibodies expressed in mouse cell cultures, from which the antibody to tetanus toxin was originally derived. In addition, the single-chain antibody accumulates to about 1% of cellular protein and appears to be quite stable within the chloroplast.

These studies indicate that pharmacologically important antibodies can be produced in algae at high levels and that these antibodies act exactly as do antibodies produced in more traditional and expensive systems. Chloroplast-produced antibodies appear to be quite stable, a situation that should allow easy purification, and algae-produced antibodies should lack many of the undesirable toxins that often contaminate antibodies expressed in bacterial cells.

Chloroplasts are the site of synthesis of many key components of plants, including lipids, amino acids, and carbohydrates. The main carbohydrate produced by plant chloroplasts is starch, a storage compound produced at high levels that humans use for a variety of purposes, including animal food and as a starting material in many organic syntheses. A number of carbohydrates of pharmacologic and commercial importance have starting material similar to that used in starch biosynthesis.

We designed a gene construct to introduce a nonplant enzyme, haluronic acid synthetase, into chloroplasts to determine if the novel carbohydrate hyaluronic acid can be produced in C reinhardtii chloroplasts. Production of this carbohydrate in microalgae could provide an important source for this scarce compound, which is used in a variety of biomedical applications, including treatment of arthritic joints.

Publications

Cohen, A., Yohn, C.B., Bruick, R., Mayfield, S.P. Translational regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Methods Enzymol. 297:92, 1998.

Kim, J., Mayfield, S.P. Protein disulfide isomerase as a regulator of chloroplast translational activation. Science 278:1954, 1997.

Mayfield, S.P., Cohen A. Translational regulation in the chloroplast. Curr. Top. Plant Physiol. 19:174, 1998.

Yohn, C., Cohen, A., Danon, A., Mayfield, S.P. A poly(A) binding protein functions in the chloroplast as a message specific translation factor. Proc. Natl. Acad. Sci. U.S.A. 95:2238, 1998.

 

 







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