Construction of protein chip to detect binding of Mitf protein (microphthalmia transcription factor) and E-box DNA.
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.
Yang
Sang-Hee
SH
Department of Biological Engineering, Skin-bioactive Material NRL, Inha University, Incheon, South Korea.
Han
Jung-Sun
JS
Baek
Seung-Hak
SH
Kwak
Eun-Young
EY
Kim
Hae Jong
HJ
Shin
Jeong-Hyun
JH
Chung
Bong-Hyun
BH
Kim
Eun-Ki
EK
D002352:Carrier Proteins / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry*; D024721:E-Box Elements; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D055767:Immobilized Proteins / Q000138:chemical synthesis; D058619:Maltose-Binding Proteins; D051739:Microphthalmia-Associated Transcription Factor / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D004269:DNA, Bacterial; D055767:Immobilized Proteins; C497234:MITF protein, human; D058619:Maltose-Binding Proteins; D051739:Microphthalmia-Associated Transcription Factor; D011993:Recombinant Fusion Proteins