Date of Graduation
Eberly College of Arts and Sciences
Knowledge of the interaction between proteins is necessary for understanding how the cellular machinery operates. Multiple methods have been developed to study protein-protein interaction, but only two have been able to provide large amount of information for S. cerevisae protein-protein network. Due to the multiple spurious data coming from these methods, there is a need of new complementary methods to improve confidence of the interactions. A new method is developed in this dissertation where the protein complexes are pulled down (immunoprecipitated) using tagged single chain antibodies specific for one of the proteins. Different affinity tags are chosen as candidates for immunoprecipitation and tested that they don't interfere with the antibody-antigen binding while remaining active and providing a clean purification in mild conditions. Strep tag II is chosen to be used as a first step in a double tag immunoprecipitation process, followed by purification using SV5 tag. Immunoprecipitation using the described method is proved in a model system. Multiple single chain antibodies are selected against proteins from a real system, Shewanella oneidensis MR-1, and checked for specificity and diversity. These single chains are used in an immunoprecipitation experiment.
Valero Aracama, Maria Rosa, "Development of a tagged scFv based immunoprecipitation method for protein -protein interaction studies" (2007). Graduate Theses, Dissertations, and Problem Reports. 4346.