Article

Searching for DNA-protein interactions by lambda phage display.

Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Fondazione Istituto Pasteur-Cenci Bolognetti, Università La Sapienza, 00161, Rome, Italy.
Journal of Molecular Biology (impact factor: 4). 10/2002; 322(4):697-706.
Source: PubMed

ABSTRACT We applied phage display technology to DNA-protein interaction studies. A cDNA expression library displayed on the surface of bacteriophage lambda was generated from the highly differentiated MMH E14 murine hepatic cell line. Selection of this library using the promoter sequence of the liver-enriched transcription factor HNF1alpha gene as ligate identified DNA-binding domains specifically interacting with different regions of this regulatory sequence. One of the selected phage showed 100% identity to a DNA-binding domain shared by differentiation specific element-binding protein, vasoactive intestinal peptide receptor-repressor protein and replication factor C and was further investigated. Specific binding of the selected protein domain was confirmed in a phage-independent context. By combining ELISA and South-Western assays using the selected phage and a bacterially expressed glutathione-S-transferase protein fused to the encoded DNA-binding domain, an array of multiple adjacent DNA-binding sites sharing a common consensus motif was identified. The strategy described represents a powerful tool to identify proteins that bind to DNA regulatory elements.

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Keywords

bacteriophage lambda
 
cDNA expression library
 
common consensus motif
 
different regions
 
differentiated MMH E14 murine hepatic cell line
 
differentiation specific element-binding protein
 
DNA-protein interaction studies
 
liver-enriched transcription factor HNF1alpha gene
 
phage display technology
 
phage-independent context
 
powerful tool
 
selected phage
 
South-Western assays
 
vasoactive intestinal peptide receptor-repressor protein