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Development of a Highly Active Fluorescence-Based Detector for Yeast G Protein-Coupled Receptor Ste2p

  • Hong, Jin Woo (School of Biotechnology, Yeungnam University) ;
  • Ahn, Hee Jun (School of Biotechnology, Yeungnam University) ;
  • Baek, Jee Su (School of Biotechnology, Yeungnam University) ;
  • Hong, Eun young (School of Biotechnology, Yeungnam University) ;
  • Jin, Dong Hoon (Asan Institute for Life Science, Department of Convergence Medicine, College of Medicine, University of Ulsan) ;
  • Khang, Yong Ho (School of Biotechnology, Yeungnam University) ;
  • Hong, Nam Joo (School of Biotechnology, Yeungnam University)
  • Received : 2018.05.02
  • Accepted : 2018.09.17
  • Published : 2018.10.28

Abstract

Twenty analogs of $[Orn^6,D-Ala^9]{\alpha}-factor$ were synthesized and assayed for their biological activities: seven analogs of $[Orn^6,X^9]{\alpha}-factor$, seven analogs of $[X^6,D-Ala^9]{\alpha}-factor$, five analogs of $[X^5,X^6,D-Ala^9]{\alpha}-factor$, and native ${\alpha}-factor$ (X = amino acids). Their biological activities (halo, gene induction, and affinity) were measured using S. cerevisiae Y7925 and LM102 and compared with those of native ${\alpha}-factor$ (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU-STE2 vector. $[Dap^6,D-Ala^9]{\alpha}-factor$ with weak halo activity (10%) showed the highest receptor affinity (> 230%) and the highest gene induction activity (167%). $[Arg^6,D-Ala^9]{\alpha}-factor$ showed the highest halo activity (2,000%). The number of active binding sites per cell (about 20,000 for strain LM102) was determined using a newly-designed fluorescence-based detector, $[Arg^6,D-Ala^9]{\alpha}-factor-Edan$, with high sensitivity (12,500-fold higher than the absorption-based detector $[Orn^6]{\alpha}-factor-[Cys]_3$).

Keywords

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