| Summary: | 碩士 === 國立中正大學 === 分子生物研究所 === 91 === 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). Phosphorylation of Ser-67 by Cdk-5 decreases the ability of PKA to phosphorylate inhibitor-1. Inhibitor-1β, one of the isoforms of human inhibitor-1, contains 132 amino acids of which the NH2-terminal 61 amino acid sequences are identical to the wild-type while the sequence after residue-61 is significantly different. 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 Chelating-Sepharose chromatography. A peptide in which the sequence based on residues 112 to 130 of inhibitor-1β was conjugated with thioglobulin. The conjugated peptide served as an antigen to induce antibody. The purified protein and antibody can be used to study the biochemical function of inhibitor-1β.
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