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KMID : 0545120180280101589
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Journal of Microbiology and Biotechnology
2018 Volume.28 No. 10 p.1589 ~ p.1603
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Development of a Highly Active Fluorescence-Based Detector for Yeast G Protein-Coupled Receptor Ste2p
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Hong Jin-Woo
Ahn Hee-Jun
Baek Jee-Su
Hong Eun-Young
Jin Dong-Hoon
Khang Yong-Ho
Hong Nam-Joo
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Abstract
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Twenty analogs of [Orn6,D-Ala9]α-factor were synthesized and assayed for their biological activities: seven analogs of [Orn6,X9]α-factor, seven analogs of [X6,D-Ala9]α-factor, five analogs of [X5,X6,D-Ala9]α-factor, and native α-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 α-factor (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU-STE2 vector. [Dap6,D-Ala9]α-factor with weak halo activity (10%) showed the highest receptor affinity (> 230%) and the highest gene induction activity (167%). [Arg6,D-Ala9]α-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 newlydesigned fluorescence-based detector, [Arg6,D-Ala9]α-factor-Edan, with high sensitivity (12,500-fold higher than the absorption-based detector [Orn6]α-factor-[Cys]3).
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KEYWORD
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a-factor, fluorescence detector, affinity, LM102
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