Directed immobilization of DNA-binding proteins on a cognate DNA-modified chip surface.
Here we describe a useful method for the site-directed immobilization of proteins with a DNA-binding domain (DNA-BD) on the cognate DNA-coated gold surface for surface plasmon resonance (SPR) imaging analyses. In order to assess the performance of this procedure, we utilized two DNA-BDs, yeast GAL4 DNA-BD, and bacterial LexA DNA-BD. After the immobilization of the cognate double-stranded DNAs (dsDNAs) to a gold chip surface with a monolayer of poly(l-lysine) for sequence-specific DNA-protein interaction, purified recombinant GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP fusion proteins were applied to a dsDNA-spotted gold chip, and were subsequently analyzed using an SPR imaging system. Consequently, the recombinant DNA-binding proteins, GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP, were shown to bind selectively to their cognate DNA sequences on the gold chip. Collectively, our results revealed that sequence-specific dsDNA microarray approach could prove useful in performing the site-directed immobilization of DNA-binding proteins onto a gold thin film in a parallel format, and thereby potentially allowing for the analysis of transcription factor binding profiling as well as for the monitoring of protein-protein interactions between target proteins with DNA-binding domain as a fusion tag and their binding partners.
Jeong
Eun-Ju
EJ
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea.
Jeong
Yoo Seok
YS
Park
Kyoungsook
K
Yi
So Yeon
SY
Ahn
Junhyoung
J
Chung
Sang J
SJ
Kim
Moonil
M
Chung
Bong Hyun
BH
D001665:Binding Sites; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*
D020099:Coated Materials, Biocompatible; D004268:DNA-Binding Proteins; D004247:DNA