Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities.
Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins.
Department of Life Science, National Dong Hwa University, Hualien County 974, Taiwan, ROC.
Yi-Jyun
YJ
Jheng
Wei-Yi
WY
Tsai
Kuo-Hsuan
KH
Chen
Kuo-Wei
KW
Lin
Chia-Lin
CL
Chyan
Ching-Chi
CC
Yang
Kuo-Chih
KC
Lin
D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D001713:Biphenyl Compounds / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D027742:Dioscorea / Q000737:chemistry / Q000235:genetics*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D007155:Immunologic Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D051379:Mice; D010587:Phagocytosis / Q000187:drug effects; D010851:Picrates / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000276:immunology
D000975:Antioxidants; D001713:Biphenyl Compounds; D004220:Disulfides; D007155:Immunologic Factors; D010851:Picrates; D010940:Plant Proteins; D011994:Recombinant Proteins; D051197:Toll-Like Receptor 4; C095057:dioscorin protein, Dioscorea; C004931:1,1-diphenyl-2-picrylhydrazyl