Design and Optimization of SPR Based Binding Assay for Evaluation and Screening of MIRF-E-Box Binding Inhibitor

Title
Design and Optimization of SPR Based Binding Assay for Evaluation and Screening of MIRF-E-Box Binding Inhibitor
Authors
김은기
Keywords
MITFm SPR Depigmentation MBP? MITF E-box
Issue Date
2013
Publisher
MOLECULAR BIOTECHNOLOGY
Series/Report no.
MOLECULAR BIOTECHNOLOGY; vol.53 no.3 startpage 265 endpage 273
Abstract
Melanin synthesis is a complex phenomenon which involves about 192 known gene products. Among them, MITF is a key transcription factor for tyrosinase, Trp1 and Trp2 proteins, which are essential for melanin biosynthesis. Thus, intervening inhibitor for the MITF?Ebox complex formation can downregulate melanin synthesis. The focus of the present study is to develop a surface plasmon resonance-based system to screen the MITF? E-box complex inhibitor. The standardization of the MITF and E-box binding assay was calibrated for kinetics and specificity, in the presence of a pre-incubated 22 mer sequence containing mutated E-box (CTTGAG) along MITF. The binding assay with C17 was optimized and the steady-state kinetics was evaluated. C17 was identified as inhibitor to MITF?E-box, by virtual screening followed by in vitro assessment and EMSA assay. The ka and kd were found to be 5.5 9 103 M-1 s-1 and 0.0014 s-1, respectively, while the steady-state association constant (KA) was 3.928 9 106 M-1. The resonance variations after inhibition were quantified and analyzed to develop the standard method for screening of microphthalmia transcription factor? E-box binding inhibitor.
URI
http://dspace.inha.ac.kr/handle/10505/31991
ISSN
1073-6085
Appears in Collections:
College of Engineering(공과대학) > Biotechnology (생명공학) > Journal Papers, Reports(생명공학 논문, 보고서)
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