TY - JOUR
T1 - One-step purification and structural characterization of a recombinant His-tag 11S globulin expressed in transgenic tobacco.
AU - Valdez-Ortiz, Angel
AU - Rascón-Cruz, Quintín
AU - Medina-Godoy, Sergio
AU - Sinagawa-García, Sugey R.
AU - Valverde-González, María E.
AU - Paredes-López, Octavio
N1 - Funding Information:
The authors would like to thank Dr. F. Guevara-Lara, from CINVESTAV-IPN, for discussion and reviewing this paper. The partial financial support from CONACYT-Mexico is acknowledged as well.
PY - 2005/2/23
Y1 - 2005/2/23
N2 - 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 (pI) of 5.58 and a basic polypeptide (21 kDa) with pI 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.
AB - 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 (pI) of 5.58 and a basic polypeptide (21 kDa) with pI 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.
UR - http://www.scopus.com/inward/record.url?scp=18444379694&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2004.09.013
DO - 10.1016/j.jbiotec.2004.09.013
M3 - Artículo
C2 - 15639103
SN - 0168-1656
VL - 115
SP - 413
EP - 423
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 4
ER -