Structural and Functional Study of PGI Complexed with 5PAH Cloning, Expression, Purification and Crystallographic Study of JAB1

• Main Authors: Jing-Meei Lin, 林淨美
• Other Authors: Chwan-Deng Hsiao
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/84223007692788345774

碩士 === 台北醫學院 === 細胞及分子生物研究所 === 90 === Structural and Functional Study of PGI Complexed with 5PAH Abstract Phosphoglucose isomeraes (PGI) catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The PGI structures of Human、rabbit and Bacillus stearothermophilus have been published at high solution. Those provide evidences to understand the molecular mechanism of PGI. However the data show that the phosphate group of substrates binding to PGI has different orientation. There is unclear the role of residues at catalyze site. In addition, PGI has been show to have functions equivalent to neurolenkin, autocrine motility factor, and maturation factor. Here, we study the X-ray crystal structure of Bacillus stearothermophilus PGI complexed with 5PAH (5-phosphospho-D- arabinohydroxamate ), A potent chemo-therapeutic agent that mimics the high energy intermediate product of PGI. 5PAH is also the best inhibitor of the isomerization reaction reported to data with a ki of 1×10-7 M. The PGI-5PAH complex structure has been determinate at 2.3 angstron resolution. It is belong to orthorhombic lattice. The space group is I222. The phase was determinate by molecular replacement method. The position of 5PAH in the enzyme active site predict the residues of Arg202、Glu285、Lys420 and His306 from the another subunit were involved in the PGI reaction proceeds. Cloning, Expression, Purification and Crystallographic Study of JAB1 Abstract JAB1 (Jun activating binding protein 1) enhances the transcriptional activity of c-Jun and JunD homodimeric complexes by stabilizing them on their cognate AP-1 (activating protein 1) DNA binding sites. JAB1 is also known as COP9 signalosome subunit 5 (CSN5), which is a component of the COP9 signalosome regulatory complex (CSN). The complex is essential for both plant and animal developments. Recently, JAB1 has been identified to associate with numbers of diverse target proteins that play roles in many cell processes, including the regulation of the JNK-mediated MAP kinase pathway, nuclear hormone signaling, and cell cycle progression. In this study, the JAB1wt and JAB1 mutant (ΔN101) of mouse musculus have been cloned into the NusA-fusion expression vector and overexpressed in Escherichia coli. The high quantity of the pure wild type JAB1 and its mutant have been obtained using a His-Tag affinity column. But it can’t obtain the JAB1 along after the restriction protease digestion. The crystallization trial of both fusion proteins has been initiated by the hanging- and the sitting-drop vapor-diffusion methods. There have microcrystals of JAB1-NusA fusion protein need to modify. The crystal structure of JAB1 and its mutant will provide valuable insight into the molecular mechanism of JAB1 recognition of the target proteins.