Environmental Biotechnology

Bioinformatics and Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 13 No. 1, Issue of January 15, 2010
© 2010 by Pontificia Universidad Católica de Valparaíso -- Chile Received July 2, 2009 / Accepted September 30, 2009
DOI: 10.2225/vol13-issue1-fulltext-3

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
Research Institute for Bioresources
Okayama University

Takafumi Kouno¹
Research Institute for Bioresources
Okayama University

Bunichi Ezaki*
Research Institute for Bioresources
Okayama University
E-mail: bezaki@rib.okayama-u.ac.jp

*Corresponding author

Financial support: Ministry of Education, Culture, Sports, Science and Technology of Japan [(Grant-in-Aid for Scientific Research for Postdoc fellowship No. 16-04467 to Y.K. and B.E.) and (Grant-in-Aid for Scientific Research (C)(2) No. 16580046 to B.E.)] and JSPS Joint Project under Japan-Korea Cooperative Science Program to B.E.

Keywords: AtGST11 gene, biopanning, DNA binding proteins, T7 phage differential display, transcription factors.

Present address: #Indonesia Institute of Science Cibinong Science Centre JI Raya Bogor Km. 46 Cibinong 16911, Indonesia.

¹These two authors contributed equally as first authors in this study.


cDNA: complementary DNA
DIG: digoxigenin
MS medium: Murashige and Skoog medium
PCR: polymerase chain reaction
SA-PMP: streptavidin paramagnetic particle
SSC: 150 mM NaCl, 15 mM sodium citrate buffer (pH 7.0)

Abstract   Full Text

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.

Supported by UNESCO / MIRCEN network