Heterologous expression of plant glycosyltransferases for biochemistry and structural biology.

Autor:	Prabhakar PK; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States; Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oakridge, TN, United States., Wang HT; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States; Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oakridge, TN, United States., Smith PJ; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States; Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oakridge, TN, United States., Yang JY; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States; Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States., Barnes WJ; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States; Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States., Peña MJ; Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oakridge, TN, United States., Moremen KW; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States; Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States., Urbanowicz BR; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States; Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oakridge, TN, United States. Electronic address: breeanna@uga.edu.
Jazyk:	angličtina
Zdroj:	Methods in cell biology [Methods Cell Biol] 2020; Vol. 160, pp. 145-165. Date of Electronic Publication: 2020 Jun 13.
DOI:	10.1016/bs.mcb.2020.05.002
Abstrakt:	Much of the carbon captured by photosynthesis is converted into the polysaccharides that constitute plant cell walls. These complex macrostructures are composed of cellulose, hemicellulose, and pectins, together with small amounts of structural proteins, minerals, and in many cases lignin. Wall components assemble and interact with one another to produce dynamic structures with many capabilities, including providing mechanical support to plant structures and determining plant cell shape and size. Despite their abundance, major gaps in our knowledge of the synthesis of the building blocks of these polymers remain, largely due to ineffective methods for expression and purification of active synthetic enzymes for in vitro biochemical analyses. The hemicellulosic polysaccharide, xyloglucan, comprises up to 25% of the dry weight of primary cell walls in plants. Most of the knowledge about the glycosyltransferases (GTs) involved in the xyloglucan biosynthetic pathway has been derived from the identification and carbohydrate analysis of knockout mutants, lending little information on how the catalytic biosynthesis of xyloglucan occurs in planta. In this chapter we describe methods for the heterologous expression of plant GTs using the HEK293 expression platform. As a demonstration of the utility of this platform, nine xyloglucan-relevant GTs from three different CAZy families were evaluated, and methods for expression, purification, and construct optimization are described for biochemical and structural characterization. (© 2020 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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