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Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector
Hiroko Hagiwara1, Sumiko Kunihiro, Keiichi Nakajima
1Research Center for Glycoscience, Advanced Institute of Industrial Science and Technology, Central Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Journal of Biochemistry
|December 11, 2002
Summary
Phage display technology was adapted for genome-wide screening of DNA-binding proteins. This method successfully enriched specific DNA-binding proteins, demonstrating its potential for sequence-specific enrichment.
Area of Science:
- Molecular Biology
- Biotechnology
- Genomics
Background:
- Phage display is a powerful technique for identifying proteins that bind specific targets.
- Genome-wide screening for DNA-binding proteins is crucial for understanding gene regulation.
Purpose of the Study:
- To evaluate the potential of phage display for genome-wide screening of DNA-binding proteins.
- To develop and validate a modified lambda phage display system for this purpose.
Main Methods:
- Construction of yeast genomic libraries using novel lambda phage-based vectors.
- Affinity selection of phages using GAL4 UAS(G) as a probe.
- Enrichment and analysis of selected phage polypeptides.
Main Results:
- Enrichment of GAL4-expressing phages by approximately 5 x 10^5-fold.
- Identification of known or putative polynucleotide-binding proteins in approximately 90% of selected polypeptides.
- Demonstration of sequence-specific enrichment of DNA-binding proteins.
Conclusions:
- The modified lambda phage display vector and enrichment technique are highly effective for identifying DNA-binding proteins.
- This approach shows significant potential for large-scale, sequence-specific screening of DNA-binding proteins.
- The study validates a novel method for discovering proteins involved in DNA binding.