Expression, Purification and Characterization of Human Galectin-12

• Main Authors: Yeh, Shu-Han, 葉書涵
• Other Authors: Chou, Wei-Yuan
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/35650519477713915193

碩士 === 國防醫學院 === 生物化學研究所 === 103 === Galectin-12, one of the most important protein involved in lipolysis pathway in adipocyte. Inhibiting galectin-12 can be an efficient therapeutic strategy for the treatment of obesity. Unfortunately, understanding the protein structure of galectin-12 is severely hampered due to inefficient expression/purification of galectin-12. Here, we describe a method to purify glectin-12 with chaotropic agents. The denatured, linearized, His-tagged protein was purified using nickel column and refolded using PierceTM protein refolding kit (Thermo Scientific). Purified protein exhibits functional interaction with lactose, its specific binding partner indicating the presence of functionally active protein upon refolding. An array of PIP (Phosphatidylinositol phosphate) strip showed that galectin-12 specifically interact with all 7 PIPs. Further, an array of 156 different glycan revealed specific binding of galectin-12 for 8 glycans. In our future experiments, we will try to understand the structural properties using X-ray crystallography and using isothermal titration calorimetry (ITC) and BiacoreTM SPR system to characterize the carbohydrate-binding and lipid-binding activity of galectin-12. We believe understand the structure of galectin-12 will help us to design appropriate drug for the treatment of obesity.