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Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions

หน่วยงาน Edinburgh Research Archive, United Kingdom

รายละเอียด

ชื่อเรื่อง : Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions
นักวิจัย : Chen, Kai
คำค้น : GFP , green fluorescent protein , protein-protein , DNA restriction/modification , Time-resolved fluorescence
หน่วยงาน : Edinburgh Research Archive, United Kingdom
ผู้ร่วมงาน : Dryden, David , EaStChem
ปีพิมพ์ : 2554
อ้างอิง : http://hdl.handle.net/1842/4886
ที่มา : -
ความเชี่ยวชาญ : -
ความสัมพันธ์ : Chen, K., Roberts, G. A., Stephanou, A. S., Cooper, L. P., White, J. H., Dryden, D. T. F. (2010) Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes. Biochem. Biophys. Res. Commun. 398 254-259. , Robert K. Neely, Gintautas Tamulaitis, Kai Chen, Marta Kubala, Virginijus Siksnys and Anita C. Jones, Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes, Nucleic Acids Research, 2009, Vol. 37, No. 20 6859–6870. doi:10.1093/nar/gkp688
ขอบเขตของเนื้อหา : -
บทคัดย่อ/คำอธิบาย :

Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM system is EcoKI from Escherichia coli K12. Both bacteriophage and conjugative plasmids have developed a variety of strategies to circumvent the host RM system. One such strategy involves the production of antirestriction proteins that mimic a short segment of DNA and efficiently inhibit the RM system. The main aim of this project was to analyse the interaction of EcoKI and its cognate methylase (MTase) with the T7 antirestriction protein, known as overcome classical restriction (Ocr), and various ArdA antirestriction proteins. Currently, there is a paucity of structural data on the complex formed between the Type I system and the antirestriction proteins. The aim of this work was twofold; (i) compare the interaction of MTase with DNA and Ocr and (ii) quantify the strength of interaction between MTase and various ArdA proteins. The MTase was fused to the Green Fluorescent Protein (GFP) to facilitate determination of the orientation of interaction with DNA and Ocr. Time resolved fluorescence measurements were carried out using the GFP-MTase fusion to determine the fluorescence lifetime and anisotropy decay. These experiments were conducted using a time resolved fluorescence instrument fabricated in-house. The values determined in these experiments were then used to perform fluorescence resonance energy transfer (FRET) measurements with fluorescently labelled DNA or Ocr. These measurements gave information concerning the relative orientation of the MTase with either DNA or Ocr. The GFP-MTase fusion was also used to quantify the strength of interaction with various ArdA proteins. Previous attempts to determine the strength of interaction between MTase and ArdA proteins by employing conventional techniques have been unsuccessful. Therefore, a novel method was developed that exploits the interaction of MTase with a cation exchange medium, which can subsequently be displaced upon binding to ArdA. This method facilitated the determination, for the first time, of a set of binding affinities for the MTase and ArdA interaction.

บรรณานุกรม :
Chen, Kai . (2554). Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions.
    กรุงเทพมหานคร : Edinburgh Research Archive, United Kingdom .
Chen, Kai . 2554. "Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions".
    กรุงเทพมหานคร : Edinburgh Research Archive, United Kingdom .
Chen, Kai . "Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions."
    กรุงเทพมหานคร : Edinburgh Research Archive, United Kingdom , 2554. Print.
Chen, Kai . Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions. กรุงเทพมหานคร : Edinburgh Research Archive, United Kingdom ; 2554.