Studies on the Biochemical and Structural Characteristics of Recombinant Rice Sucrose Synthase RSuS3 in Pichia pastoris

• Main Authors: YI-CHUN TSAI, 蔡逸君
• Other Authors: 王愛玉
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/14841930544789354814

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 94 === Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. Among the three isogenes in rice (RSus1, RSus2, RSus3), the gene products of RSus1 and RSus2 are ubiquitously present in all rice tissues, while the expression of RSus3 is seed-specific. In this research, recombinant RSuS3 expressed from Pichia pastoris is used to study on the biological and structural characteristics of RSuS3. Expression plasmid containing RSus3 cDNA transformed into yeast P. pastoris for expression, and the recombinant product is purified to analyze its properties. The results show that the major activity of recombinant RSuS3 is to generate sucrose from fructose and UDPG, while the sucrose-cleavage activity of RSuS3 is very low. Besides, Mg2+ is able to promote the sucrose-synthesis activity of RSuS3. The presence of sugar metabolites have no significant effect on recombinant RSuS3 activity, neither do the presence of phospholipids. There are eight cysteines on the surface of recombinant RSuS3, and the native protein is present as a tetramer as revealed from the result of transmission electron microscopy (TEM). Furthermore, native crystal, heavy atom co-crystal and diffraction data of native crystal at 3.53 Å resolution has been obtained as the initial results of first step of X-ray crystallography.