Binding of Arabidopsis thaliana SSIII-SBD to glycogen, starch and its components

ALOMAR, M. L.; WAYLLACE, N. Z.; PALOPOLI, N.; VALDEZ, H.; UGALDE, R.; PARISI, G.; DIEGO FABIAN GOMEZ CASATI; BUSI, M. V.

Rosario

Congreso; XLII Reunión SAIB; 2006

The starch-synthase III (SSIII, At1g11720), with a total of 1025 residues, is one of the enzymes involved in plant starch synthesis. SSIII from Arabidopsis thaliana contains a putative N-terminal transit peptide followed by a 557-amino acid SSIII specific domain with three internal repeats and a C-terminal catalytic domain of 450 amino acids. Using computational characterization techniques, we showed that each of the three internal repeats encodes a starch-binding domain (SBD). To address whether the different SSIII-SBDs are able to bind to polysaccharides, we cloned and expressed in E. coli cells His-tagged proteins with three (SBD-full), two (SBD-int) or one (SBD-small) SBD domains. The three proteins were purified to near homogeneity by one-step purification using a Ni++ column. SDS-PAGE and western blot analysis revealed the presence of three protein bands of 66, 34 and 15 kDa for each SBD in agreement with the predicted molecular mass. We tested the ability of each SBD to bind to glycogen, amylose and  amylopectin using a co-sedimentation assay. Our results showed that each SBD could bind to each polymer with different affinites.