| Project/Area Number |
11450315
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
生物・生体工学
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
MUROOKA Yoshikatsu Osaka Univ. Eng. Professor, 大学院・情報科学研究科, 教授 (60029882)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
HIRAYAMA Noriaki Tokai Univ. Eng. Professor, 大学院・医学研究科, 教授 (70238393)
ONO Hisayo Osaka Univ. Eng. Assis. Prof., 遺伝情報実験センター, 助手 (40224274)
YAMASHITA Mitsuo Osaka Univ. Eng. Assoc. Prof., 大学院・工学研究科, 助教授 (40220347)
|
|---|
| Project Period (FY) |
1999 – 2002
|
| Project Status |
Completed (Fiscal Year 2002)
|
| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1999: ¥7,400,000 (Direct Cost: ¥7,400,000)
|
|---|
| Keywords | metallothionein / heavy metal binding protein / Cd / Zn / molecular design / DNA binding / Arsenic |
|---|
| Research Abstract |
Mammalian metallothionein (MT) binds 7 divalent metal ions to its 20 cysteines. It has a role as a cellular reservoir for zinc and in distribution of zinc and is although thought to participate in detoxification of heavy metals and in antioxidant defenses. MT is a suitable model for investigating molecular interactions relating to the handling of metals in cells. However, the production of functional MT proteins in microoganisms has been limited because of the instability of MT. The supply of MT for research, therefore, continues to rely on its purification from mammalian tissue, in particular because rather large quantities can be prepared from livers of animals in which MT has been induced by cadmium.
I) We developed the overproduction system for MT as an intein fusion protein, purified by affinity chromatography, cleaved proteolytically by incubation with DTT, and prepared by reconstitution of thionein with cadmium or zinc.
2) To increase the binding ability and to stabilize MT, we designed genes for dimeric and tetrameric MTs and the genes were successfully overexpressed in Escherichia coli to generate functional oligomeric MTs.
3) Arsenic is a toxic element that is found in the atmosphere, as well as in quantic and terrestrial environments. We have demonstrated that more than 6 gram atoms of AS^<3+> bind one molecule of human MT by UV absorption spectroscopy, ICP-AES, and MALDI-TOF-MS.
4) We have constructed a new metal-binding site in the human MT-2, using the protein as a scaffold to investigate the structure and function of metal-binding. To evaluate the metal-binding affinity of the mutant proteins, we performed pH titrations of wild-type and mutant proteins.
5) We found that wild-type and mutant MTs bound DNA but the two MTs showed different affinity of binding to DNA.
|
