Genetically design and synthesis of multifunctional fusion proteins for biosensor applications

The key to the development of biosensors, which have gained importance in recent years, is to create a biocompatible biosensor interface by preserving the biological activity of the molecules required for proper sensor performance. The biomolecules used to form the interface are difficult to immobilize on the surface of electrodes or other inorganic materials, especially due to the sensitive nature of enzymes, which are functional proteins, and their tendency to denature quickly. The immobilization of the protein provides superior stability, separation and reusability compared to the use of free protein. However, classical methods for protein immobilization have adverse effects on the function of proteins. In this study, a one-step, easy, controlled, oriented and directed immobilization was performed using the peptide that fully adheres to the silica surface to eliminate the undesirable effects of protein immobilization. Multifunctional protein (MBP-SiBP) designed by molecular cloning methods was synthesized in bacteria by expression vector containing Maltose Binding Protein (MBP) and purified by affinity chromatography. The designed protein was characterized by advanced analysis methods such as protein determination methods, electrophoresis techniques and mass spectrophotometer. Functional comparison of MBP-SiBP multifunctional protein with each other was performed using peptide-free MBP (wt-MBP) as a control. Due to the self-assembly feature of the silica-binding peptide, it has been proven by Micro-contact printing method that MBP-SiBP protein provides controlled immobilization without the need for any chemical or physical modification. It was observed that MBP-SiBP multifunctional protein did not lose its functions during immobilization as it interacted actively with fluorescent bound antibody and maltose.