Molecular Cloning and Characterization of the Tomato Cystatin Expressed in E. coli

• Main Authors: Wei-Hung Chi, 紀緯鴻
• Other Authors: Min-Ying Wang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/76705338668107576150

碩士 === 國立中興大學 === 生物科技學研究所 === 95 === Tomato (Solanum lycopersicum ) leaves induced by methyl jasmonate produce a cystatin similar to potato multicystatin. Tomato and potato cystatins possess the characteristics of high molecular weight and eight repeat cystatin domains. Tomato cystatin which displays similar biochemical properties to PMC is limited to protein level studies. This study focuses on determination of tomato multicystatin (TMC) gene sequence by RT-PCR and 5’RACE extension methods, the relationship between TMC and PMC and comparison of two recombinant TMC activities expressed in E. coli. The result shows that the full-length cDNA of TMC gene is 2339 bp in size encoding 754 amino acid residues. The calculated PI value in all of TMC eight domains is between 4.9 to 8.8. It consists exclusively of eight repeat cystatin domains, with 57% to 84%, which consists of four conserved regions. Expression of domain1 (TMCD1) and domain1&2 (TMCD1&2) recombinant TMCs affect the growth of E. coli. Both expressed TMCD1 and TMCD1&2 proteins exhibited papain inhibitory activity. In conclusion, it is shown high similarity of TMC and PMC in deduced amino acid sequences. Expressed TMCD1 and TMCD1&2 displayed inhibitory activities against a cysteine proteinase.