| Budget Amount *help |
¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000)
Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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| Outline of Final Research Achievements |
We established recombinant expression systems for extracellular domains of mouse Nectin-1 and mouse Nectin-3 in insect cell Sf-9. First we tried to purify Nectin-1 / Nectin-3 heterodimer fraction from the mixture of cultural supernatants. However, the result of surface plasmon resonance analysis indicated the fast dissociation rate constant of heterophilic interaction between Nectin-1 and Nectin-3, suggested difficulty of stable hetero-complex purification. Thus we next established individual purification systems for Nectin-1 and Nectin-3, respectively. The purified Nectins were mixed and used for crystallization experiments. As a result, structures of Nectin-1 homodimer and Nectin-3 homodimer were obtained from mixture of purified Nectin-1 and Nectin-3 in 2.24 A and 2.58 A resolutions, respectively (PDB ID: 5B21and 5B22). Additionally, chemical crosslink experiment using BS3 crosslinker revealed that the minimal unit of Nectin-1 / Nectin-3 heterophilic complex is heterodimer.
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