Development of a Highly Active Fluorescence-Based Detector for Yeast G Protein-Coupled Receptor Ste2p.
J Microbiol Biotechnol
; 28(10): 1589-1603, 2018 Oct 28.
Article
em En
| MEDLINE
| ID: mdl-30441882
ABSTRACT
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 (ï¼ 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 newly-designed 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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MEDLINE
Assunto principal:
Proteínas de Saccharomyces cerevisiae
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Receptores Acoplados a Proteínas G
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Fator de Acasalamento
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article