Poster: Protein targeting & vesicular trafficking

Abs # 663: One-step purification and structural characterization of recombinant His-tag amarantin expressed in transgenic tobacco

Presenter:	Valdez-Ortiz, Angel
Authors	Valdez-Ortiz, Angel  (A)   Medina-Godoy, Sergio  (A)   Valverde-Gonzáles, Maria E.  (A)   Paredes-López, Octavio  (A)
Affiliations:	(A): CINVESTAV-IPN, Unidad Irapuato

Amarantin is one of the most important storage proteins of amaranth seeds, with relevant nutritional-functional and nutritional characteristics. Amarantin cDNA was cloned in-frame with a sequence enconding a polyhistidine tag and expressed from a 35S promoter in transgenic tobacco seeds. The presence of a (His)"_"6"" tag on the polypeptide permitted rapid and high-yield single-step purification using immobilized metal-ion affinity chromatography (0.5-5% of purified His-tag protein of total soluble protein seeds). Biochemical characterization indicated that purified His-tag amarantin migrated with the expected molecular weight and was correctly processed into acidic and basic subunits linked by a disulfide bridge, moreover it was assembly in both homo- and hetero-hexameric 11S structures. The strategy presented here was designed to have a high-yield expression and purification procedure for this important protein and should prove useful to do structural-functional studies. Results shown here are the first report manipulating the amino acidic sequence of amarantin so it would aid in its further specific modification.