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

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Title
Construction of protein chip to detect binding of mitf protein (microphthalmia transcription factor) and E-box DNA
Author(s)
S H Yang; J S Han; S H Baek; E Y Kwak; H J Kim; J H Shin; Bong Hyun Chung; E K Kim
Bibliographic Citation
Applied Biochemistry and Biotechnology, vol. 151, no. 2, pp. 273-282
Publication Year
2008
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.
Keyword
E-boxHTSMitfProtein chip
ISSN
0273-2289
Publisher
Springer
DOI
http://dx.doi.org/10.1007/s12010-008-8186-3
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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