Purification of Recombinant Xylosyltransferase XXT2 involved in Xyloglucan Biosynthesis

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2014-04-15
Authors
Young, Zachary
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Biochemistry, Biophysics and Molecular Biology
Abstract

Plant cell walls are important in society as food, fuel, and fiber. However, many aspects of cell wall biosynthesis remain unknown. The polysaccharide xyloglucan plays an important role in the structure of the primary cell wall of dicotyledonous plants and is the most abundant hemicellulose in dicots. Xyloglucan biosynthesis involves at least seven different type II transmembrane glycosyl transferases in the Golgi. The goal of this research is to determine the conditions to successfully express and purify the soluble fully-folded recombinant XXT2, a critical xylosyltransferase involved in xyloglucan biosynthesis. Using E. Coli to express the protein with an attached tag, affinity and size-exclusion chromatography was used to purify the protein, then it was digested to remove the tag from the protein. To confirm the activity and the correct conformation of the enzyme, HPLC and MALDI-TOF were used. By expressing and purifying the folded, soluble proteins, protein-protein interactions will be investigated, the residues involved in these interactions will be determined, and also proteins can be prepared for structural characterization using X-ray crystallography. This research will help to understand the polysaccharide biosynthesis, and also assist in the plant cell wall modifications to improve biomass properties improvements for industrial applications.

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