Applying the Bioinformatic Methods to Design and Evaluate the SapM-M13 pIX Fusion Protein and Its Theoretical Role in the Phage ELISA System

Abstract

Phage ELISA is a common method used to confirm binding of obtained phages from phage display technique to related antigens. Enzyme-conjugated antibody directed against the major capsid protein (pVIII) or enzyme-conjugated secondary antibody against the primary antibody is used as a detection system in phage ELISA. We suggested expression of the secreted acid phosphatase (SapM) enzyme on M13 pIX minor coat protein directly, and evaluated this hypothesis using <em>In Silico </em>techniques. 3D structure model of the fusion protein (SapM+M13 pIX) was generated and evaluated by related software. MD simulation and TMHMM program results showed a stable fusion protein which is anchored to the inner membrane of <em>E. col </em>by membrane spanning region suggesting aproper assembling on M13 phage. In theory, SapM enzyme on the phage surface can catalyze the p-nitrophenyl phosphate as substrate and creates yellow color which can be measured at OD=405 nm by microtiter plate reader. We believe that decreasing the antibody layers in phage ELISA will significantly increase the reliability and reproducibility of the test and reduce its time.