One-step purification and structural characterization of a recombinant His-tag 11S globulin expressed in transgenic tobacco.
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.
Valdez-Ortiz
Angel
A
Depto. de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 629, 36500 Irapuato, Gto., Mexico.
Rascón-Cruz
Quintín
Q
Medina-Godoy
Sergio
S
Sinagawa-García
Sugey R
SR
Valverde-González
María E
ME
Paredes-López
Octavio
O
D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004220:Disulfides / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005916:Globulins / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D007526:Isoelectric Point; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000737:chemistry / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*; D014461:Ultracentrifugation
D018076:DNA, Complementary; D004220:Disulfides; D005916:Globulins; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D006639:Histidine