Identification and characterization of human inhibitor-1β

• Main Author: 蘇鈴珠
• Other Authors: 黃憲斌
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/58514459302811011726

碩士 === 國立中正大學 === 分子生物研究所 === 90 === Inhibitor-1 is an acid- and thermostable protein. Phosphorylation of Thr-35 by cAMP-dependent protein kinase (PKA) converts inhibitor-1 to a potent and specific inhibitor of protein phosphatase-1 (PP1). Inhibitor-1β, one of the isoforms of human inhibitor-1, contains 132 amino acids of which the N-terminal 61 amino acid sequences are identical to the wild-type while the sequence after residue-61 is significantly different from the wild-type. In this study, I subcloned the cDNA of inhibitor-1β into the pET-32a plasmid, and the vector was expressed in E. coli BL21 (DE3). The product from the expression vector contained a fusion tag (159 amino acids) and inhibitor-1β. I purified the fusion inhibitor-1β to 95% homogeneity by Q-Sepharose, Blue-Sepharose and gel filtration chromatography. The fusion inhibitor-1β was treated with thrombin to get inhibitor-1β that contains a small piece of sequence (30 amino acids). After PKA phosphorylation, I found that phospho-inhibitor-1β was a potent inhibitor of PP1. The results suggested that inhibitor-1β could mimic the biological function of inhibitor-1 in regulation of PP1 activity.