A phage display combined with DNA affinity magnetic system can be applied to a screening of DNA binding proteins, such as transcription factors

• Kusumadewi Sri Yulita ^[1] ; Takafumi Kouno ^[1] ; Bunichi Ezaki ^[1]
  1. [1] Okayama University

     Okayama University

     Naka-ku, Japón

     
• Localización: Electronic Journal of Biotechnology, ISSN-e 0717-3458, Vol. 13, Nº. 1, 2010, págs. 14-15
• Idioma: inglés
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• Resumen

  • Here we introduce a new approach for the screening of DNA binding proteins, using a phage library based on a phage display technique. In principal, a complementary DNA (cDNA) library based on the recombinant bacteriophage T7 expressing target proteins on its capsid (phage display) is constructed. These phage particles are hybridized with a biotinylated target DNA fragment which is immobilized on the surface of streptavidin paramagnetic particle (SA-PMP). The phage particles are released from the target DNA fragment by a nuclease treatment and the recovered phages are used to the next round of hybridization. These processes are repeated three times to amplify the target phages in the population. This simple method is faster, and more systemic than other current methods (e.g. yeast one hybrid system). As a proof of this principle, we tried to isolate transcription factors which specifically bind to the promoter region of the Arabidopsis thaliana AtGST11 gene. Two obtained candidates, RING zinc finger protein and AtHB6, showed DNA binding activity to the AtGST11 promoter region. We could validate that our new application of phage display is a superior method for isolation of DNA binding proteins with a broad range of potential applications.