| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2013: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2012: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Outline of Final Research Achievements |
Since pyrrolysly-tRNA synthetase (PylRS)/pyrrolysine tRNA (tRNA(Pyl)) pair is orthogonal to aminoacyl-tRNA synthetase/tRNA pairs in various organisms, PylRS/tRNA(Pyl) pair has been used as one of the parental molecules to cotranslatinally incorporate an unnatural amino acid into a protein. In this project, I tried to develop the system to produce a biotinylated protein in E. coli by using PylRS/tRNA(Pyl) pair and biotinyl-L-lysine (biocitin). Although several challenges of in vivo selection to PylRS libraries with saturated mutations, many PylRS mutants recognizing natural amino acid were isolated as higher background. In addition to the selection, I constructed the PylRS mutant based on the docking model of PylRS and biocitin, which however showed the activity with very little. These results suggest that large modification of the active site in PylRS is required to obtain PylRS mutant that can charge biocitin onto tRNA(Pyl).
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