Affinity selection of DNA-binding proteins displayed on bacteriophage lambda.
Two transcription factors, human ATF1, its DNA-binding domain (ATF1BD), and the DNA-binding domain (GAL4BD) of the yeast GAL4 protein, were displayed on the surface of bacteriophage lambda vectors and efficiently selected by DNA fragments immobilized in microtiter wells. The DNA-binding proteins are fused to the carboxy terminus of the tail protein gpV and head protein gpD of the vectors, lambdafoo and lambdafooDc, respectively. After a single round of affinity selection, the fusion phages were successfully enriched 60- to 4,000-fold over the vector phages. Further, the GAL4BD fusion phages were enriched 5- and 15-fold by affinity selection using specific DNA as probes over nonspecific DNA when expressed on lambdafooDc and lambdafoo, respectively. The ATF1BD fusion phages were also sequence-specifically enriched greater than 4-fold when displayed on lambdafoo. These results suggest that the lambdafoo display system is useful for in vitro studying of protein-DNA interactions and may be applied to screening of DNA-binding protein from complex cDNA libraries through DNA-binding affinity.
Zhang
Y
Y
Department of Molecular Biology, National Institute of Bioscience and Human Technology, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Pak
J W
JW
Maruyama
I N
IN
Machida
M
M
D051697:Activating Transcription Factor 1; D010582:Bacteriophage lambda / Q000235:genetics*; D001710:Biotin / Q000031:analogs & derivatives* / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics; D005822:Genetic Vectors; D015345:Oligonucleotide Probes / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000032:analysis* / Q000235:genetics
D051697:Activating Transcription Factor 1; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C093051:biotin-SS-oligonucleotide; D001710:Biotin