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Title 

Construction of protein chip to detect binding of mitf protein (microphthalmia transcription factor) and E-box DNA

Authors 

S H YangJ S HanS H BaekE Y KwakH J KimJ H ShinBong Hyun ChungE K Kim

Publisher 

Humana Press

Issue Date 

2008

Citation 

Applied Biochemistry and Biotechnology, vol. 151, no. 2, pp. 273-282

Keywords 

bacterial DNAcarrier proteinhybrid proteinimmobilized proteinmaltose binding proteinmaltose-binding proteinmicrophthalmia associated transcription factorMITF protein, humanchemistryE box element

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 β-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 μM) and Mitf (up to 500 μg/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.

ISSN 

0273-2289

Link 

http://dx.doi.org/10.1007/s12010-008-8186-3

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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