TY - JOUR
T1 - Expression and characterization of the acidic subunit from 11S Amaranth seed protein
AU - Luna-Suárez, Silvia
AU - Medina-Godoy, Sergio
AU - Cruz-Henández, Andrés
AU - Paredes-López, Octavio
PY - 2008/2
Y1 - 2008/2
N2 - 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-β-D-thiogalactoside, at 37°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.
AB - 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-β-D-thiogalactoside, at 37°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.
KW - 11S acidic subunit
KW - Amaranth globulin
KW - E. coli-expressed protein
KW - Seed storage protein
UR - http://www.scopus.com/inward/record.url?scp=41049096761&partnerID=8YFLogxK
U2 - 10.1002/biot.200700146
DO - 10.1002/biot.200700146
M3 - Artículo
C2 - 18034435
AN - SCOPUS:41049096761
SN - 1860-6768
VL - 3
SP - 209
EP - 219
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 2
ER -