Purification and Crystallization of the Chemosensory Protein of Forcipomyia taiwana

• Main Authors: Sheng-Chuen Yang, 楊詩純
• Other Authors: Chao-tsang Lu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/7nn9r9

碩士 === 中臺科技大學 === 醫學生物科技研究所 === 96 === Forcipomyia taiwana is a blood-sucking insect, which is found in Taichung County by Japanese entomologist Siraki in 1913, and has the serious influence in Taiwan area in recent years. The place bitten by it may cause rash, intense pruritus, swelling, and even allergy. In the previously study, F. taiwana has the chemosensory protein (CSP) in chemosensory organs. CSP has been identified in the several sense organs and could bind the hydrophobic molecular in the environment, such as CO2, fatty acid and pheromone, then affects the olfactory receptors and cause a succession of chemical signal transmission. So, CSP influences insect’s reproduction and the behavior of looking for food, and it acts the quite important role to the insect’s multiplied. In this research, we want to explore the tertiary structure of CSP of F. taiwana and look for suitable ligand to interfere the information transmission and reduction bite on the human. In the experiment, we expressed the gene of CSP of F. taiwana in Kluyveromyces lactis, and obtained the high-purity CSP protein after a series process of ammonium precipitation, cation-exchange and gel filtration chromatography, and demonstrated that CSP had not post-translation modification by LC/MS/MS. Then, we expressed the gene of CSP of F. taiwana in Escherichia coli, and obtained the high-purity CSP protein by the same purified method. By further analyzing its secondary structure and functionality, we found CSP of F. taiwana is mainly composed by α-helix and it has activeness. We combined the CSP of F. taiwana with different ligands, and used the hanging drop vapor diffusion method to sieve out the crystal generated conditions, and then we found star shaped crystals in the 23th condition of the commercialized reagent “Crystallization Basic”. But those crystals are too small to assay by X-ray diffraction method.