2014 Fiscal Year Annual Research Report
| Project/Area Number |
13F03026
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| Research Institution | The University of Tokyo |
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Principal Investigator |
菅 裕明 東京大学, 理学(系)研究科(研究院), 教授 (00361668)
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| Co-Investigator(Kenkyū-buntansha) |
JONGKEES Seino 東京大学, 理学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2013-04-01 – 2016-03-31
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| Keywords | 人口触媒 / 糖転移 / ペルチド / 翻訳 |
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| Outline of Annual Research Achievements |
In my project, I aim to select catalytic peptides capable of forming or breaking glycosidic bonds. I have successfully synthesised a set of glycosylated haemagglutinin-tag peptides with an N-terminal alkyne residue. The reaction between this alkyne and an azide in peptides expressed in a cell-free translation system was optimised, and a test selection using this validated the intended selection strategy. Recovery of non-glycosylated haemagglutinin tag-linked peptides was at least 100 fold higher than any of the glycosylated forms. All of these glycosylated peptides gave similar recovery to a negative control with no added haemagglutinin tag peptide. A second strategy, for formation of glycosidic bonds, is also planned, with the required biotinylated glycosyl donor reagent being synthesised following a literature method.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Progress towards selection of catalytic carbohydrate-modifying peptides is so far very good, with all reagents required to start the selection now ready. The strategy for selection of glycoside-cleaving catalysts has been shown to give both affinity to the selection resin and good discrimination between the starting material and product forms, while the selection for a glycosidic-bond forming catalyst involves covalent biotinylation, and so is a robust and well-known strategy for selection. A set of three mRNA libraries has also been assembled successfully, based on a modification of a literature method, giving a range of peptide sizes that can be selected using the above-described reagents. This diversity of strategies and libraries will hopefully ensure that the selection process is successful.
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| Strategy for Future Research Activity |
The haemagglutinin tag peptides are being resynthesised to take advantage of copper-free strain-promoted cycloaddition chemistry, which is almost complete. This will remove the need for purification steps during the selection process, improving both recovery and workflow. Following completion of this synthesis and subsequent purification of the peptides, selection can commence. This will be carried out with two different strategies and three libraries of varying peptide length and architecture. Increasing stringency and decreasing reaction time will be used across multiple rounds, with intermittent error-prone PCR to increase diversity and allow convergence on active peptides that may not have been represented in the initial library. Experiments will then be carried out to validate the reaction catalysed, whether multiple turnover is possible, and eventually investigate the mechanism of catalysis.
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