원문정보
초록
영어
A protein chip was developed for melanogenesis inhibitor screening. MITF (Microphthalmia-associated transcription factor) is a key regulatory transcriptional factor of the pigmentation-related genes such as tyrosinase and MC1R, and binds to specific sequence (CATGTG) in E-box DNA within the promoter of tyrosinase and MC1R in the melanocytes.1), 2) We produced MITF as a MBP (maltose binding protein) fusion protein in E. coli and purified by affinity column and immobilized on β-cyclodextrin coated glass plate. Binding of MITF to its target DNA, E-box oligomer, was monitored by surface plasmon resonance (SPR) and SPR imaging (SPRi).
Unlike SPR and SPRi which employed unlabeled DNA oligomer, we used Cy3-labeled DNA in fluorescent imaging.3) In this method, fluorescent intensity was proportional to the DNA concentration up to 20μM. Among these detection methods, fluorescence method was the most reliable.4) Kinetics of DNA binding with MITF showed Langmuir isotherm and its kinetic
constants were determined. To determine if interaction between MITF and E-box is sequence specific, we modified E-box DNA (CATGTG → CAGCTG) and monitored fluorescent intensity. These results indicated the potentials of MITF-E-box DNA chip as a screening tool for depigmenting agent in the cosmetic industry.