Luna-Suárez Silvia, Medina-Godoy Sergio, Cruz-Hernández Andrés, Paredes-López Octavio
Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados de IPN, Unidad Irapuato, Irapuato, Guanajuato, México.
Biotechnol J. 2008 Feb;3(2):209-19. doi: 10.1002/biot.200700146.
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.
苦马豆素酸性亚基有潜力作为一种功能性和营养性蛋白质被利用。为了评估这两种可能性，该蛋白质在携带表达质粒pET - AC6His的重组大肠杆菌Origami (DE3)中产生。测定了三个不同的表达因素：诱导剂浓度、温度和苦马豆素酸性亚基积累的时间。结果表明，0.3 mmol/L浓度的异丙基 - β - D - 硫代半乳糖苷、37℃以及诱导后6小时有利于苦马豆素酸性亚基的高表达，且大多以包涵体形式存在。通过固定化金属亲和色谱从可溶性部分纯化该蛋白质，每升 terrific 肉汤培养物可获得高达30 mg的苦马豆素酸性亚基。对表达的可溶性苦马豆素酸性亚基进行蔗糖密度梯度超速离心分析表明，它组装成单体。苦马豆素酸性亚基的表达以及一步纯化将有助于通过定点诱变对这种贮藏蛋白进行进一步研究。
