| 研究課題/領域番号 |
20K20197
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| 研究機関 | 北陸先端科学技術大学院大学 |
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研究代表者 |
Rajan Robin 北陸先端科学技術大学院大学, 先端科学技術研究科, 助教 (70848043)
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| 研究期間 (年度) |
2020-04-01 – 2023-03-31
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| キーワード | Protein therapeutics / Zwitterionic Polymer / Glycopolymers / ultracentrifugation / Micelles / RAFT polymerization |
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| 研究実績の概要 |
In this year, I have developed a novel micellar system made of poly-sulfobetaine (capable of protecting proteins from denaturation) and trehalose (well-known for protecting proteins and cells from damage). Self-assembly is induced by the addition of hydrophobic polycaprolactone. The polymers were prepared using a one-pot reversible addition-fragmentation chain transfer (RAFT) polymerization, which allowed us to control the chain length as well as other polymer parameters. The three components work synergistically to produce micelles with a remarkable ability to protect different proteins under several types of stress (high temperature and multiple freeze-thaw cycles). Protein stability studies were carried out using lactate dehydrogenase (LDH) and recombinant human insulin under several types of stress. Additionally, proteins retain their enzymatic activity and higher-order structure in the presence of these micelles. Importantly, the micelles can be easily removed from the proteins by a simple ultracentrifugation step, and the recovered proteins retain their native activity and secondary structure. This clearly demonstrates that this micellar system not only protects proteins but can easily be separated from the protein sample whenever required, enabling the protein to be employed for various applications without additives.
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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
I have been able to successfully complete a major part of the project this year by synthesizing and optimizing the conditions for the preparation of the zwitterionic micellar system. The micellar system was tested against different proteins under different conditions and they could effectively protect proteins as well as facilitate them to retain their native structure and property. Additionally, I developed a method to separate proteins from the micelle by a simple centrifugation step. This allowed us to not protect the proteins, but at the same time, isolate the proteins in their native state.
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| 今後の研究の推進方策 |
As part of the future work, I will test the efficiency of micelles to protect more complicated as well as more relevant systems such as monoclonal antibodies. Additionally, I will test the efficacy of zwitterionic polymers to protect mRNA and other therapeutic proteins, which are currently being developed as vaccine candidates for combating infectious diseases. Moreover, I will try to elucidate the mechanism of protection by these zwitterionic polymers as well as investigate (qualitatively as well as quantitatively) the kind of interaction existing between the polymer and proteins by various methods like isothermal titration calorimetry, molecular simulation, and nuclear magnetic spectroscopy.
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| 次年度使用額が生じた理由 |
A lot of time was spent on optimizing the conditions and developing newer systems and methods for preparing as well as testing protein stability. Hence, not much funding was required. However, in the current year, I intend on employing lots of proteins, antibodies, and many other therapeutic molecules. Additionally, I will investigate the interaction of proteins with polymers, which would require the purchase of many chemicals as well as the need to travel to other research institutes to carry out certain experiments. Moreover, due to the current pandemic, it was not possible to attend workshops and conferences to showcase the work, however, I expect to travel in this year in order to discuss the research work with a wider scientific community and get some feedback.
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