Development of novel proteins as a defence against potential biothreat agents

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DESCRIPTION (provided by applicant): The ongoing war on terror has created the necessity of a whole new spectrum of systems that are able to detect and inactivate biothreat agents, systems that are inexpensive to produce, robust and stable to allow their use on a massive scale by first responders or even the general population. However, properties of antibodies that are currently used as main components in such systems do not allow their prolonged storage without refrigeration and simple incorporation into electronic devices. Also, antibodies are expensive to produce and when used as antidotes cannot inactivate agents that have been already internalized into the cells. Here, we propose to convert a small and extremely stable domain of bacterial protein into an entity capable of specific binding and inactivation of biothreat agents. We expect that the resulting product will be inexpensive to produce, easy to modify to allow its adaptation to the needs of specific protocols and will be stable enough to survive harsh environmental conditions and even transport through the cell membrane barrier. In Phase I, we will test the feasibility of our approach by developing proteins that will recognize the catalytic domain of botulinum neurotoxin serotype A (BoNT/A-L). For this, we will create several types of phage displays of the chosen bacterial protein and screen them for the ability to recognize a specially created derivative of BoNT/A-L. Also, during Phase I, we will create a system that will allow extremely sensitive monitoring of enzymatic activity of BoNT/A-L. This system will be used to monitor the efficiency of the screening process and to determine affinities of selected constructs. During Phase II, we will prove that the developed protocol allows development of proteins recognizing any biothreat agent of interest. For this, we will apply the protocol to the evolution of proteins capable of recognizing and/or neutralizing botulinum neurotoxins of serotypes B and C.