Heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the plant cell wall. Here, we report a detailed, and reproducible method of purification of catalytic domain of CESA1 from Arabidopsis thaliana that was recombinantly expressed in Escherichia coli. The method relies on a two stage purification procedure to obtain the catalytic domain in monomer and trimer forms. The biochemical and biophysical data including low resolution structures of the protein have been published (Vandavasi et al., 2016). Currently the crystallization studies of this protein are underway.
[Background] Cellulose is the most important structural component of plant cell walls and constitutes the Earth’s largest source of biorenewable material, yet the mechanism of its synthesis by plants is poorly understood. The plant cellulose synthesis complex (CSC), also called a ‘rosette’ because of its hexameric appearance in electron microscope images, is a large multi-subunit transmembrane protein complex responsible for synthesis of cellulose chains and their assembly into microfibrils. The number of cellulose synthase (CESA) proteins in the CSC and the number of cellulose chains in a microfibril have been debated for many years. Structural information about CESA proteins from plants is crucial to provide answers to some of the basic questions regarding the mechanism of cellulose synthesis. However, elucidation of the structure of CESA proteins has proved difficult because they are multi-domain proteins comprised of disordered, globular, and membrane associated domains. As an alternative to pursuing structural studies of CESA holoproteins, we are developing approaches for recombinant expression of individual CESA domains (e.g., N-terminal domain, central-cytosolic domain, C-terminal transmembrane domain) in large quantities suitable for structural studies. The current protocol has been optimized for isolation of the catalytic domain of A. thaliana CESA1 as reported (Vandavasi et al., 2016). Using this protocol, it is possible to control the oligomerization state of the protein enabling structural studies of the monomer and the trimeric form of the protein. The approach described may be broadly applicable to other systems.
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