IDENTIFICATION OF AFFIBODY MOLECULES THAT TARGET CROTALID VENOM AND TARGETED PROTEIN IDENTIFICATION
Collom, Clancy Kate
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The Crotalinae subfamily of pit vipers, which includes rattlesnakes, copperheads, and water moccasins, are commonly found in many parts of the U. S., including Texas. CroFab® and AnaVip®, the current antivenoms for pit viper snake bites in the U.S., are derived from antibody fragments harvested from sheep and horses and effectively treat most snake bites, but there are some limitations to the antivenoms, including allergic reactions. In order to expand our knowledge of snake venoms and possibly open the door to new antivenom options, an affibody phage display library was used to identify affibody molecules that specifically target Crotalid snake venom proteins. By working with the library, two affibody molecules were found to bind to the venoms of three varieties of rattlesnakes, Crotalus atrox, Crotalus scutulatus, and Crotalus viridis. The venom protein targets of the affibody molecules are unknown. The purpose of this project is to identify the protein targets of the affibody molecules using a pull down assay followed by mass spectrometry analysis. The affibodies were identified while attached to the M13 bacteriophage protein pIII. For further characterization, we cloned them into an expression vector for production and purification. The affibodies were cloned into a p6xHis-SNAP-tag plasmid. A nickel affinity column was used to purify the affibody. Then the SNAP-tag labeled affibody was covalently bound to SNAP-capture magnetic beads, and a solution of snake venom was washed over the bound affibody. The affibody bound its target in the snake venom, and unbound snake venom proteins were washed away.