研究実績の概要 |
The goal of this research is the identification of macrocyclic-peptide ligands that can be used as agonist or antagonists for a specific signaling pathway. For the selection of macrocyclic peptides that bind to BMP signaling pathway receptors, the in vitro translation system used for the production of macrocyclic peptides was reconstituted with some modifications. The in vitro translation system was made from a combination of commercial products and in-lab prepared reagents, which is simpler than the method used by the Suga Lab, where all components are produced in-lab, and will hopefully make this technology more accessible to other academic labs. Using clones from previous selections, we established that macrocyclic peptides could be produced in the University of Tsukuba using a combination of commercial products from New England Biolabs and biomolecules prepared in our lab, namely the ribozyme flexizyme. Accurate production of the intended macrocyclic peptide was confirmed using MALDI analysis. In addition, the production of a linear peptide library and a corresponding macrocyclic-peptide library was also produced and verified. The in vitro selection target, bone morphogenetic protein (BMP) receptor ALK1, was received from my collaborator, Dr. Wei Li, as a histidine-tagged DsbA fusion protein. Binding of the target to magnetic beads was confirmed. The typical in vitro selection for macrocyclic peptides usually requires 6 rounds of selection. I am currently on the fourth round of selection.
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現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The in vitro selection for macrocyclic peptides that bind to ALK1 receptor is underway. My collaborators are still preparing the remaining selection targets, type 2 receptors BMPR2 and ActR2. I have just completed the fourth round of selection for ALK1-binding macrocyclic peptides. In round 1 to round 3 of selection, we expect little indication that the selection is moving forward smoothly. At the current stage in my previous selections, round 4, an increase in recovered cDNA, indicating the isolation of functional macrocyclic peptides, is usually observed. However, I have yet to see this increase. While this does not indicate that the selection is not proceeding, close examination of the library at this stage is warranted. Toward this goal, the gene pools of various rounds have been translated into peptides and are awaiting examination using MALDI analysis. The mass of dominant clones will be observable above the background signal of the random peptide library if the selection is proceeding correctly. Furthermore, the individual sequences can be identified using next-generation sequencing. Selections against other protein targets are being performed in parallel, and these serve as controls for the anti-ALK1 selection. These controls will facilitate the identification of any systematic error in the system.
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今後の研究の推進方策 |
We are currently troubleshooting the ALK1 selection using previously selected clones from successful selections performed in the past. Specifically, the macrocyclic peptide clone MaL6 will be used in a binding assay against PfMATE. This and other systems identified in the Suga Lab at the University of Tokyo are reliable and will help identify any problems in the selection.
Upon optimization of the selection system using MaL6:PfMATE, we can restart the selection against ALK1 from any round if a complete restart is deemed unnecessary. Identification of the functional anti-ALK1 macrocyclic peptides will be performed using next-generation sequencing. Identified macrocyclic peptides will then be chemically synthesized and purified prior to being assayed for ALK1 binding and for inhibition of BMP signaling. We have already purchased an HPLC for the purification of the synthesized peptides. We are currently looking at several options for peptide synthesizers, with a preference for Biotage due to its ability to synthesize peptides in parallel. In addition, we are also planning on purchasing a rotary evaporator and a lyophilizer for drying the purified peptides.
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