Preparation of A Monoclonal Antibody that Recognizes Hepp1

• Main Authors: Yi-Da Li, 李易達
• Other Authors: Hsien-Bin Huang
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/67882152567352011028

碩士 === 國立中正大學 === 生物醫學研究所 === 104 === Phosphoprotein phosphatase (PPP) is one of the protein phosphatases. The most well studied enzymes in PPP family are PP1, PP2A and PP2B. In cells, the catalytic subunit of PP1 is associated with the different regulatory subunits that target the enzyme to different subcellular compartments where PP1 modulates the functions of its substrates through dephosphorylation. Up to now, more than 200 PP1-regulatory proteins have been identified. Heart-specific protein phosphatase 1-binding protein (Hepp1) is one of PP1-regulatory proteins found within human heart cells in our laboratory by using yeast two-hybrid screen. We identified that Hepp1 only exists in human myocardium. In this study, I prepared the recombinant Hepp1 and the monoclonal antibody that recognizes Hepp1. The Hepp1 monoclonal antibody will be applied to identify Hepp1 in human myocardium in the future. I transformed a plasmid (pET32a-Trx-Ub-Hepp1) into E. coli BL21 (DE3). The transformed E. coli was cultured in a LB Broth medium at 37℃. Then, the Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the expression of Trx-Ub-Hepp1 fusion protein. The Trx-Ub-Hepp1 fusion protein was purified by the Ni2+-Sepharose and SP-Sepharose chromatographies. The Hepp1 unit could be cleaved out from the fusion protein by yeast ubiquitin C-terminal hydrolase-1(YUH-1) and purified by SP-Sepharose chromatography. The purified recombinant Hepp1 was sent to the Kelowna company for preparation of the anti-Hepp1 monoclonal antibody. The Hepp1 monoclonal antibody can effectively recognize the recombinant Hepp1 by western blot. Keywords: PP1, Hepp1, monoclonal antibody