Expression and characterization of the acidic subunit from llS Amaranth seed protein

Abstract

Amarantin acidic subunit has the potential to be employed as a functional and a nutraceutical pro­ tein_ To evaluate both possibilities this protein was produced in recombinant Esdlerichia coli Origa­ mi (DE3) harboring the expression plasmid pET-AC6His. Three different expression factors were assayed: inductor concentration, temperature and time of the amarantin acidic subunit accumu­ lation . The results indicated that a 0.3 mmolfl concentration of isopropyl- o-thiogalactoside. at 3rc and 6 h after induction were favorable for high expression of amarantin acidic subunit, most­ ly in the form of indusion bodies. The protein was purifoed from soluble fraction by immobilized metal affinity chromatography, up to 30 mg amarantin acidic subunit/L Terrifoc broth culture were obtained. Sucrose density gradient ultracentrifugation analysis of the expressed soluble amaran­ tin 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.