2P090 In vitro evolution of Melittin using a liposome display(01F. Protein: Engineering,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Abstract

Biomolecules such as protein, peptide and nucleic acids interact with each other in the cell controlling its life activities. Therefore, techniques to reproduce biomolecular interactions correctly on the solid substrate would be a good versatile way to clarify the biological phenomena. In this research, we investigated the influence of protein orientation and density on peptide-protein interaction (adopting cysteine synthase (CS) complex formation as the model) detection with employing site specific protein immobilization technique. As the results, consideration of the location of functional site with avoiding the steric hindrance by adjusting the protein density and the degree of freedom of the peptide would be important to design higher interaction detection system. We aim to develop a new strategy of protein design based on the phage display and single-molecule sorting. In the first step of the method, a library of phages expressing a target protein with randomized sequence is prepared. In the second step, the expressed proteins are labeled by fluorophore(s) and are sorted based on the fluorescence signals from single phages. To realize the strategy, we prepared a phage expressing C-terminal fragment (214-230) of GFP. The addition of the N terminal fragment (1-214) of GFP, purified separately, to the phage caused the reconstitution of GFP and the appearance of the fluorescence signal. We are currently conducting the observation and sorting of the phages having the reconstituted GFP on the surface. 2P086 無機基板表面を標的としたラクダ抗体から着想するスマート なバイオセンサー仕様抗体の設計