Expression and characterization of the acidic subunit from 11S Amaranth seed protein.
Amarantin acidic subunit has the potential to be employed as a functional and a nutraceutical protein. To evaluate both possibilities this protein was produced in recombinant Escherichia coli Origami (DE3) harboring the expression plasmid pET-AC6His. Three different expression factors were assayed: inductor concentration, temperature and time of the amarantin acidic subunit accumulation. The results indicated that a 0.3 mmol/L concentration of isopropyl-beta-D-thiogalactoside, at 37 degrees C and 6 h after induction were favorable for high expression of amarantin acidic subunit, mostly in the form of inclusion bodies. The protein was purified from soluble fraction by immobilized metal affinity chromatography, up to 30 mg amarantin acidic subunit/L Terrific broth culture were obtained. Sucrose density gradient ultracentrifugation analysis of the expressed soluble amarantin acidic subunit revealed that it was assembled in monomers. The expression of the amarantin acidic subunit, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.
Luna-Suárez
Silvia
S
Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados de IPN, Unidad Irapuato, Irapuato, Guanajuato, México.
Medina-Godoy
Sergio
S
Cruz-Hernández
Andrés
A
Paredes-López
Octavio
O
D027721:Amaranthus / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D013058:Mass Spectrometry; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism*
D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins