Expression and purification of intact and functional soybean (Glycine max) seed ferritin complex in Escherichia coli.
Soybean seed ferritin is essential for human iron supplementation and iron deficiency anemia prevention because it contains abundant bioavailable iron and is frequently consumed in the human diet. However, it is poorly understood in regards its several properties, such as iron mineralization, subunit assembly, and protein folding. To address these issues, we decided to prepare the soybean seed ferritin complex via a recombinant DNA approach. In this paper, we report a rapid and simple Escherichia coli expression system to produce the soybean seed ferritin complex. In this system, two subunits of soybean seed ferritin, H-2 and H-1, were encoded in a single plasmid, and optimal expression was achieved by additionally coexpressing a team of molecular chaperones, trigger factor and GroEL-GroES. The His-tagged ferritin complex was purified by Ni2+ affinity chromatography, and an intact ferritin complex was obtained following His-tagged enterokinase (His-EK) digestion. The purified ferritin complex synthesized in E. coli demonstrated some reported features of its native counterpart from soybean seed, including an apparent molecular weight, multimeric assembly, and iron uptake activity. We believe that the strategy described in this paper may be of general utility in producing other recombinant plant ferritins built up from two types of subunits.
Dong
Xiangbai
X
Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing 210093, People's Republic of China.
Tang
Bo
B
Li
Jie
J
Xu
Qian
Q
Fang
Shentong
S
Hua
Zichun
Z
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D012995:Solubility; D030262:Soybean Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D021122:Protein Subunits; D011994:Recombinant Proteins; D030262:Soybean Proteins; D005293:Ferritins; D007501:Iron