Searching for DNA-protein interactions by lambda phage display.
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.
Cicchini
Carla
C
Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Fondazione Istituto Pasteur-Cenci Bolognetti, Università La Sapienza, 00161, Rome, Italy.
Ansuini
Helenia
H
Amicone
Laura
L
Alonzi
Tonino
T
Nicosia
Alfredo
A
Cortese
Riccardo
R
Tripodi
Marco
M
Luzzago
Alessandra
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D010582:Bacteriophage lambda; D001665:Binding Sites; D002460:Cell Line; D002874:Chromosome Mapping; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D025741:HMGA1a Protein / Q000378:metabolism; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins; D019151:Peptide Library; D010455:Peptides; D011401:Promoter Regions, Genetic; D051818:Replication Protein C; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; C496496:Hnf1b protein, mouse; D009687:Nuclear Proteins; D019151:Peptide Library; D010455:Peptides; D012097:Repressor Proteins; C095385:Rfc1 protein, mouse; C113967:Rfc1 protein, rat; D014157:Transcription Factors; C063552:transcription factor BTF3; D025741:HMGA1a Protein; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA; D000251:Adenosine Triphosphatases; D051818:Replication Protein C; C119652:SRCAP protein, human