| Summary: | 碩士 === 中台醫護技術學院 === 醫學生物科技研究所 === 94 === RIM2 (Rab3-interacting molecule 2) is a protein of presynaptic active zone, which derived from mouse brain, and it could interact with receptor of postsynaptic active zone to adjust the release of neurotransmitter. Also, RIM2 is composed of many domains, which includes Zn2+, PDZ, C2A, C2B domains. All these domains could interact with proteins, and adjust the release of neurotransmitter. Almost all PDZ domains interact with other proteins by recognizing four residue radicals on the C-terminal of other proteins, and they could be classified into 3 kinds, according to different recognized sequences. But for PDZ domain of RIM2, it does not belong to any of these 3 kinds, although it can also recognize the four residue radicals on the C-terminal of other proteins. So, it is more special than other PDZ domains. In this research, we use X-ray to irradiate the crystal of RIM2 PDZ domain, and check its three-dimensional structure to compare with other 3 kinds of PDZ domains, and study the differences between all of them. To design this experiment, we cloned the gene of PDZ domain first, and its molecular weight will be around 20 kDa. And, purified it by the processes of deposition of ammonium sulfate, dialysis, ion exchange and gel filtration. Then, we got PDZ domain protein with high degree of purity. Also, we used hanging drop vapor diffusion method as our crystal method. By using the reagent of Crystal Screen™, which is from Hampton Research, we crystallized the high purified protein of PDZ domain with modified the condition of Crystal Screen™ 23, and found there is crystal generated. But, it is not reproduce. On the other side, we observed the drops almost show clear or cloud form, and we expect it is because the concentration of protein is too low. On this research, besides considering more how to get more and purer protein from the processes of purified method, we also need to increase the concentration of protein and sourcing any condition to make crystal of protein.
keywords: RIM2 (Rab3-interacting molecule 2)； presynaptic active zone； PDZ domain； neurotransmitter； hanging drop vapor diffusion method
|
|---|
