One-step purification and structural characterization of a recombinant His-tag 11S globulin expressed in transgenic tobacco

• Published in: Journal of biotechnology Vol. 115; no. 4; pp. 413 - 423
• Main Authors: Valdez-Ortiz, Angel, Rascón-Cruz, Quintín, Medina-Godoy, Sergio, Sinagawa-García, Sugey R., Valverde-González, María E., Paredes-López, Octavio
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 23.02.2005; Amsterdam Elsevier; New York, NY
• Subjects: 11S globulin; Acidic and basic polypeptides; Amaranth; Amaranthus hypochondriacus; Biological and medical sciences; Biotechnology; Blotting, Western; Chromatography, Affinity; Cloning, Molecular; Disulfides - chemistry; DNA, Complementary; Electrophoresis, Gel, Two-Dimensional; Fundamental and applied biological sciences. Psychology; gene expression; globulins; Globulins - chemistry; Globulins - genetics; His-tag amarantin; Histidine - chemistry; Histidine - genetics; Histidine - isolation & purification; Isoelectric Point; Molecular Weight; Nicotiana; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana tabacum; Peptides - chemistry; Plant Proteins - chemistry; Plant Proteins - genetics; Plants, Genetically Modified; Promoter Regions, Genetic; Protein Engineering - methods; Protein purification; purification; recombinant proteins; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Seeds - chemistry; Seeds - genetics; transgenes; transgenic plants; Transgenic tobacco; Ultracentrifugation; Amaranth; Amaranthus hypochondriacus; Protein purification; 11S globulin; His-tag amarantin; Transgenic tobacco; Acidic and basic polypeptides; Characterization; Purification; Transgenic plant; Globulin
• DOI: 10.1016/j.jbiotec.2004.09.013
• ISSN: 0168-1656

Amarantin, an 11S globulin, is one of the most important storage proteins of amaranth seeds, with relevant nutritional–functional and nutraceutical characteristics. Its cDNA was cloned in-frame with a sequence encoding a polyhistidine tag and expressed under the direction of a 35S promoter in transgenic tobacco seeds. The presence of a (His) 6 tag on the polypeptide permitted a high-yield single-step purification using immobilized metal-ion affinity chromatography and rapid characterization. Purified His-tag amarantin accounted for up to 5% of total soluble seed protein. Biochemical characterization indicated that purified His-tag amarantin migrated with the expected molecular weight (53 kDa) and was correctly processed into an acidic polypeptide (32 kDa) with isoelectric point (p I) of 5.58 and a basic polypeptide (21 kDa) with p I of 9.24, linked by a disulfide bridge. Moreover, His-tag amarantin was assembled into both homo- and hetero-hexameric 11S structures. These results show that the His tag did not change the biochemical and physicochemical properties of amarantin. The strategy presented here for rapid and high-yield expression and purification procedure should facilitate structure–function studies for this nutritional protein.