Functional Analysis of a Cysteine Protease from Sesamum indicum

• Main Authors: Jheng, Cing-Hua, 鄭慶華
• Other Authors: Douglas J. H. Shyu, Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/16297756872407693393

碩士 === 國立屏東科技大學 === 生物科技系所 === 104 === Cysteine proteases are known to play essential roles not only in plant growth and development but also in senescence and programmed cell death. Cysteine proteases are postulated to be responsible for the degradation and mobilization of storage proteins in seeds. Plant cysteine proteases such as papain and stem bromelain are extensively used for medicine, brewing wine, and food industry. Sesame seeds are rich in oil, proteins, unsaturated fatty acids, vitamins, minerals, and folic acid, and the major constitutions are lipids and proteins. A cysteine protease cDNA (SiCP) was isolated previously from germinating sesame seeds. Full-length of SiCP57 cDNA is 1,492 bp in length, and it contains a 1,113 bp open reading frame encoding a SiCP protein precursor of 371 amino acids. The signal peptide sequence is composed of 20 amino acids. Removal of the signal peptide sequence of SiCP cDNA is 1,056 bp in length encoding a mature SiCP protein of 351 amino acids. SiCP cDNA is cloned to an expression vector pET28a, and then the recombinant plasmid is introduced into the Escherichia coli for protein expression. The recombinant soluble protein with a size of 41 kDa is isolated from bacterial cells, and the zymographic staining does not show protease activity. SiCP57 cDNA is also cloned to expression vectors pPICZA and pGAPZA for induced and constitutively expression of recombinant protein expression, respectively. The results indicate that the recombinant proteins are not expressed in both conditions. SiCP cDNA is then cloned to an expression vector pPICZαB, and then the recombinant plasmid is introduced into the yeast Pichia pastoris for protein expression. The secreted recombinant SiCP is a soluble protein with a size of 51 kDa isolated from culture medium, and the zymographic staining demonstrated that it exhibits protease activity. The recombinant SiCP also exhibit casein and Nα-Benzoyl-DL-arginine-2-naphthylamide hydrochloride hydrolytic activities. The SiCP soluble protein is gathered by Ni-NTA resin, but the zymographic staining does not show protease activity. It was reported that nickel ions could inhibit cysteine protease activity. Therefore, other purification methods could be used to explore the optimal conditions of enzyme activity for future application in the industry.