Generation of a De Novo Actin Pointed-End Binding Protein

Abstract

In order to create disease specific drugs, molecules that are designed to bind to any given target with high affinity and high specificity have been the motivation of the biotechnology field for some time. The natural immune system can produce antibodies that essentially recognize any antigenic target with high affinity and specificity. Therefore, employing this specificity through antibody related technologies should yield bioengineered proteins that are very useful to biomedical research. We performed a high-throughput phage display screen, which used restricted amino acid diversity phage display libraries to generate antibody fragments to F-actin. We generated synthetic actin-binding antibody fragments (sABs) that were further selected to bind to specific actin conformations. These sABs resemble the Fab fragment of an antibody, with a 55kDa heterodimer of heavy and light chain variable fragments, forming an antigen-binding site that was engineered to bind to a specific actin conformation by specific library sorting and subsequent phage ELISA characterization. A series of these generated sABs were characterized in vitro and in vivo. We identified three interesting candidates for study. The first construct severs F-actin in vitro and in vivo. The second sAB bundles F-actin and inhibits G-actin from polymerizing. And the most interesting sAB is a pointed-end actin binding molecule. This construct shows clear affinity for the pointed-end of actin by in vitro TIRF analysis. Furthermore, this construct also inhibits in vitro polymerization of actin over time. The application of this technology to generate molecules to specific conformations of proteins for analysis will be pivotal in the bioengineering field.