| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2010: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2009: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
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| Research Abstract |
The aim of this study is to establish enzyme-mediated protein stabilization and its application inside living cells. We have focused on Sortase, a transpeptidase. Sortase recognizes LPXTG amino acid sequences and cleaves between T and G, and then ligated with N-terminal polyglycine sequences. Our strategy is that the substrate sequences, i.e., LPXTG and GGG sequences, are introduced both N- and C-terminus of the target protein. Then Sortase allows to conjugate its N- and C-terminus and produces the circular protein. EGFP were chosen as model proteins and these substrate tags appended EGFP was successfully conjugated by Soprtase and circular EGFP was obtained. Then the thermal stability of these circular proteins was significantly improved, suggesting our strategy is very useful for protein stabilization. The optimal linker design between N-terminal and C-terminal is one of the issue for expanding versatility of this method.
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