| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2013: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2012: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
|
|---|
| Research Abstract |
Stargazin is an auxiliary transmembrane subunit of the AMPA-type glutamate receptor which is responsible for fast excitatory synaptic transmission. The specific aim of this study is to establish a protocol to obtain recombinant stargazin proteins labeled with stable isotopes to pursue structural and interaction studies using nuclear magnetic resonance (NMR) spectroscopy as a principal investigation tool. We used the yeast Pichia pastoris for the recombinant expression because it has advantages to express human membrane proteins over prokaryotic cells such as E. Coli. We optimized experimental conditions, such as cell culture conditions, cell strains, cell disruption conditions, membrane protein extraction conditions from cell membranes, and finally obtained a purified recombinant stargazing with an yield of 4 mg per liter culture. The protocol established in this study will enable the NMR study of stargazin.
|
