Construction Of Eukaryotic Surface Display Based On The Baculoviral F Protein

The display of foreign proteins on the surface of viruses or cells provides a tool for the analysis and design of biomolecules and their interactions. Phage display of foreign proteins has been of great importance for over a decade and has proven to be a powerful technology. However, since it is a prokaryotic expression system, there are limitations, e.g., in glycosylation and the folding of complex proteins. Eukaryotic display makes the expression of these complex, glycosylated proteins possible. The use of Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) for surface display of foreign proteins has been demonstrated in several publications. For display it is sufficient to fuse the heterologous protein to the N-terminus of gp64, the major envelope protein on the surface of the AcMNPV, or to the gp64 transmembrane domain. The potential of this approach for the generation and screening of a eukaryotic expression library or for the production of monoclonal antibodies has also been demonstrated.Two distinct envelope fusion proteins are identified in baculovirus budded virions (BV). GP64 is the major envelope fusion protein of group I NPV BVs, which mediates low-pH-triggered membrane fusion and is necessary for efficient budding of BVs from the surface of infected cells. An unrelated type of envelope fusion protein, named F, is encoded by group II NPVs, such as Spodoptera exigua MNPV (SeMNPV) and Lymantria dispar MNPV (LdMNPV). Like GP64 homologous protein, F proteins mediate low-pH-dependent membrane fusion during BV entry. Cleavage of the F protein into two disulfide-linked subunits by a cellular convertase (furin-like) is necessary for the low-pH-triggered membrane fusion activity and viral infectivity. The baculoviruses with GP64 as the fusion protein such as AcMNPV and Bombyx mori NPV (BmNPV) have been exploited for surface display so far. In this report we describe the successful development of an alternative eukaryotic display system based on F protein of group II NPV, Helicoverpa armigera single nucleocapsid NPV (HaSNPV).HaSNPV is a major pathogen of the cotton bollworm H. armigera and has been wildly used as pesticide to control the host in China. As in other group II NPVs, no gp64-like gene is present in the HaSNPV genome, but an f homolog has been found as open reading frame (ORF) 133 (Hal33). Hal33 encodes an F protein, which ispost-translationally cleaved and that the two cleavage fragments, the 59 kD transmembrane anchored Fi domain and the 20 kD F2, remain associated by a disulfide bridge which is responsible for virus entry into the host cells and efficient virion budding. Since F protein is essential for the virus infection process, a second copy of the gene was introduced into the HaSNPV genome for manipulations and to achieve high levels of expression. As a model system, the marker gene encoding the 26kDa protein glutathione-S-transferase (GST) was used to construct two fusions with the/gene by the HaSNPV Bac-to-Bac system. One fusion, in which GST was inserted between the signal peptide and the mature protein, was found to be efficiently expressed and displayed on the budded virions.