BACULOVIRUS SURFACE DISPLAY - CONSTRUCTION AND SCREENING OF A EUKARYOTIC EPITOPE LIBRARY

The baculovirus expression system was utilized to serve as a tool for ligand selection, demonstrating the applicability of the system to the generation and screening of eukaryotic expression libraries. The HIV- 1-gp41 epitope 'ELDKWA', specific for the neutralizing human mAb 2F5, was inserted into the antigenic site B of influenza virus hemagglutini n and expressed on the surface of baculovirus infected insect cells. I n order to improve the antigenicity of the epitope within the hemagglu tinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope, This poo l of hemagglutinin genes was directly cloned into the baculovirus Ac-o mega, To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capa city of individual viral clones, Using fluorescence activated cell sor ting (FACS) we isolated a baculovirus clone displaying the epitope wit h markedly increased binding capacity out of a pool of 8000 variants i n only one sorting step. Binding properties of the identified ligand w ere examined by FAGS performing a competition assay.