Affinity selection of DNA-binding proteins displayed on bacteriophage lambda.

Y Zhang; J W Pak; I N Maruyama; M Machida

doi:10.1093/oxfordjournals.jbchem.a022698

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Affinity selection of DNA-binding proteins displayed on bacteriophage lambda.

Y Zhang·J W Pak·I N Maruyama·M Machida

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Abstract

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.

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Activating Transcription Factor 1 Bacteriophage lambda Biotin Blotting, Western Cloning, Molecular DNA-Binding Proteins Escherichia coli Fungal Proteins Genetic Vectors Oligonucleotide Probes Oligonucleotides Peptide Library Protein Structure, Tertiary Recombinant Fusion Proteins Saccharomyces cerevisiae Proteins Transcription Factors Journal Article Research Support, Non-U.S. Gov't

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Y Zhang, J W Pak, I N Maruyama, M Machida. 2000. Affinity selection of DNA-binding proteins displayed on bacteriophage lambda.. https://doi.org/10.1093/oxfordjournals.jbchem.a022698

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