Purification of Phytochelatin Synthase from Fission Yeast (Schizosaccharomyces pombe 806) and Preparation of Its Monospecific Antibody

• Main Authors: Pei-Hsiu Liao, 廖珮秀
• Other Authors: Rong-Huay Juang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/73319243064749556481

碩士 === 國立臺灣大學 === 農業化學研究所 === 91 === The heavy metal sequestration mechanism in a cell was triggered when cadmium was entered from the environment. In fission yeast, C. elegans, and plant, phytochelatin (PC) was synthesized to chelate the cadmium, and produced the Cd-PC complex, which will reduce the damage caused by heavy metals. PC is catalytically synthesized by the enzyme PC synthase (PCS), which is activated by cadmium in the cell. Wu (2000) purified the PCS from fission yeast, and established the purification procedure. Chiu (2001) followed that process, improved the assay protocol, purified the enzyme, and produced the specific antibody against PCS (12F9). Although the activity assay indicated that the purified enzyme was PCS, direct evidence is needed to prove this fact. Especially the gene sequence of PCS from fission yeast was depicted recently (Clemens et al., 1999), and served as an important criteria to verify the above question. This study further improved the purification procedure. Amicon was used to replace gel filtration in desalting, and metal chelating chromatography step was deleted. The monospecific antibody (3H8) was prepared by using a synthetic peptide as the antigen, which was selected from the PCS protein deduced from its genomic sequence. Several protein spots on the two-dimensional electrophoresis gel were stained by the antibodies, and their amino acid sequences were then determined by Edman degradation. Results showed that one of the spot contained part of the PCS sequence.