1.ArsA1 structure recognition with chloroplast TA protein 2. Protein purification and crystallization of 3α-HSD Y155F-K159A mutation in Comamonas testosterone

• Main Authors: Yu-Wang Su, 蘇育王
• Other Authors: Hsin-Yang Chang
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/vt54kr

碩士 === 國立中山大學 === 海洋生物科技暨資源學系研究所 === 107 === Tail-anchored (TA) membrane protein is safely transported to the endoplasmic reticulum (ER) membrane in mammals and yeast via the TRC40/GET3 binding factor. However, the mechanism by which the TA protein is delivered remains unclear in plants. This unique eukaryotic cell membrane transport system correctly distinguishes between different TA proteins for various organelles, including the mitochondria, chloroplast and ER TA proteins. In this experiment, we want to express the protein of green algae (C. reinhardtii) ArsA1 (GET3 homolog), and to understand how ArsA1 can accurately recognize, transport, and insert the TA proteins into the chloroplast membrane. We also want to find ways to improve ArsA1 activities during the photosynthesis pathway in C. reinhardtii. In this article, we use the purified ArsA1 overexpressed form E. coli for crystallization, which is similar to our previous experiment, and also found out the structure of ArsA1 protein. Then we want to realize what kind of TA protein that ArsA1 would bind, and then TA protein can be sent to the right place on the specific membrane of the organelles. For this purpose, we constructed two different kind of plasmids. One sequence is the ArsA1, and the other is TA proteins with his-tag on them. We did co-tranformation into the E. coli to express these two protein complex. During the purification step, his-tag in the TA protein complex is the only protein motif specifically binding to the Ni-NTA column rather than ArsA1. We wash out the protein by the imidazole and analysis the solution flowing out. If we can find the ArsA1, than we can say that there is interaction between the ArsA1 and this TA protein.