Construction of Protein Chip to Detect Binding of Mitf Protein (Microphthalmia Transcription Factor) and E-box DNA

Abstract

A protein chip was constructed to detect the binding of microphthalmia-associated transcription factor (Mitf) and E-box DNA. Mitf, a key regulatory transcriptional factor of pigmentation-related genes such as tyrosinase, binds to specific sequence (CATGTG) in E-box DNA within the promoter of tyrosinase in the melanocytes. We produced Mitf as a maltose-binding protein (MBP) fusion protein in Escherichia coli, purified it using an affinity column, and immobilized it on beta-cyclodextrin-coated glass plate. Binding of Mitf to its target DNA, E-box oligomer, was monitored by surface plasmon resonance (SPR), SPR imaging (SPRi), and fluorescence-based system. Among these detection methods, fluorescence method was the most reliable. In this method, fluorescent intensity was proportional to the DNA concentration (up to 20 microM) and Mitf (up to 500 microg/ml). Kinetics of DNA binding with Mitf showed Langmuir isotherm, and its kinetic constants were determined. It is expected that Mitf-E-box DNA chip can be used as a screening tool for depigmenting agents in the cosmetic industry.

A small molecule inhibitor of Mitf‐E‐box DNA binding and its depigmenting effect in melan‐a cells

“…We tested 27 chemicals for their inhibitory activity against Mitf‐E‐box DNA binding using the protein chip in which fluorescence intensities reflected Mitf‐E‐box DNA binding 12 . Among them, we found that Compound #17 exhibited potent inhibitory activity on Mitf‐E‐box DNA binding.…”

“…A protein chip assay was performed as in the previous report 12 . Briefly, MBP‐Mitf (0.5 mg/mL) was immobilized on the β‐CD‐treated glass slide chip.…”

“…In our previous report, we developed a protein chip to detect the binding of Mitf and E‐box DNA, and suggested the potential use of the Mitf‐E‐box DNA chip as a screening tool for depigmenting agents in the cosmetic industry 12 …”

“…In our previous report, we developed a protein chip to detect the binding of Mitf and E-box DNA, and suggested the potential use of the Mitf-E-box DNA chip as a screening tool for depigmenting agents in the cosmetic industry. 12 To discover the small molecule inhibitors of Mitf-E-box DNA binding using this protein chip, we screened 27 chemicals, which were obtained from a pharmacophore data base of 4 000 000 by virtual screening. Finally, we found that Compound #17 exhibited potent inhibitory activity against Mitf-E-box DNA binding.…”

Design and characterization of a maltose binding protein-linked growth factor for matrix engineering

“…Recently, Jung et al produced microphthalmiaassociated transcription factor (Mitf) as a maltose-binding protein (MBP) fusion protein in E. coli, purified it using an affinity column, and immobilized it on b-cyclodextrin-coated glass plate for a biochip construction (Yang et al 2008). Recently, Jung et al produced microphthalmiaassociated transcription factor (Mitf) as a maltose-binding protein (MBP) fusion protein in E. coli, purified it using an affinity column, and immobilized it on b-cyclodextrin-coated glass plate for a biochip construction (Yang et al 2008).…”