Molecular cloning and expression of asparaginyl endopeptidase, a specific protease in maturing sesame seed

• Main Authors: Hsin-Hui Hong, 洪心惠
• Other Authors: Jason T.C.Tzen
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/66284419250506411711

碩士 === 國立中興大學 === 農業生物科技學研究所 === 87 === There is increasing interest in using crop plants as bioreactors in the past decade including production of modified starches, plastics, industrial and pharmaceutical proteins such as enzymes, antibodies, and antigens. Oleosin, a unique structural protein in plant seed oil bodies, has been designed to carry foreign proteins for expression in seeds. The low density of oil bodies provides an easy method (simply done by subsequent centrifugation) to purify recombinant proteins carried by oleosins. However, the bottleneck for application of this expression system is the high cost of using a specific protease to cleave target proteins from the carrier, oleosin. This study aims to search for a specific protease in seeds to overcome the above obstacle. Seed storage proteins are disulfide-bonded and specifically cleaved into two subunits by a protease termed asparaginyl endopeptidase before their deposit into protein bodies. Using the cDNA library constructed with maturing sesame seeds, the corresponding gene of asparaginyl endopeptidase has cloned by PCR and completely sequenced. Currently, I am trying to over-express this gene in E. coli using the T7 polymerase-promoter system, to purify the expressed protein for antibody induction, and to inspect the adequate condition for the specific protease digestion of the expressed asparaginyl endopeptidase.