Monoclonal antibodies for plant cell walls
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Polysaccharide Isolation
wall fractionation
Cell walls will be isolated from Arabidopsis (roots, leaves and cultured cells), Medicago leaves, tomato fruits, and suspension-cultured sycamore, tobacco and rice cells. These walls will then be tretaed with a combination of endoglycanses and aqueous buffers to solubilize the major pectic and hemicellulosic polysaccharides (see Figure on right).

The solubilized polysacchardies will be purified using anion-exchange and size-exclusion chromatographies.

Additional polysaccharides will be obtained from other sources including the growth medium of suspension-cultured plant cells, Arabidopsis seed mucilage (a unsubstituted rhamnogalacturonan), Jojoba and Tamarind seeds (xyloglucan),

Click here for more details of the polysaccharide immunogens to be used in this project

Enzymic Fragmentation of Plant Cell Wall Polysaccharides

We will use oligosaccharide fragments generated from cell wall polysaccharides as immunogens. This will allow us to target specific structural features of a polysaccharide, so long as the conformation adopted by the oligosaccharide is similar to that present in the polysaccharide.

Oligosaccharides will be generated by selective enzymic fragmentation of wall polysaccharides. For example, endo-1,4-glucanse treatment of xylogucan generates a series of well-defined oligosaccharides. Oligosaccharides are also generated by treating de-arabinosylated rhamnogalacturonan I (RG-I) with rhamnogalacturonan hydrolase and rhamnogalacturonan lyase. RG-I backbone fragments will also be generated by treating Arabidopsis seed mucilage with these enzymes. Oligogalacturonides with different degrees of methyl esterification will be generated by endo-polygalacturoanse treatment of pectin

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Polysaccharide Isolation

Fragmentation of Polysaccharides

Coupling of Glycans to Proteins
Generating Hybridoma Cell Lines

scFv phage-display libraries

Characterizing Antibodies

Characterizing Epitope Structures

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A National Science Foundation-funded (Grant No DBI-0421683) research project at
The Complex Carbohydrate Research Center of The University of Georgia