Generation and presentation of membrane protein targets using baculovirus surface display
SUPERVISOR: REINGARD GRABHERR
Project assigned to: MONIKA WILDE
Membrane proteins play important roles in performing many cellular processes and thus are one of the most important targets in cancer therapy. Hence, these proteins are often used as targets for antibody-based therapies. However, membrane proteins are notoriously difficult to produce and study due to their hydrophobic nature and complex structures. Recently, baculovirus-mediated surface display has been used for the heterologous production of membrane proteins (Zhang et al., 2008). Baculoviruses acquire a lipid envelope by budding through the insect cell membrane. The envelope consists mainly of the virus-derived envelope protein gp64, but also cellular components, to some extent, can be detected on the virus surface. Various viral envelope proteins e.g. the influenza A virus hemagglutinin are incorporated into the baculovirions (Lu et al., 2007) However, markedly increased efficiency can be achieved when the transmembrane domain of the baculovirus envelope protein gp64 is used for display of the extracellular domain of hemagglutinin (Lai et al., 2007). Gp64 is the most prevalent protein during baculovirus infection and is favoured for incorporation into the viral envelope during budding. Gp64 null mutants have been constructed previously and shown to be suitable for pseudo-typing by incorporating e.g. vesicular stomatitis coat protein (Mangor et al., 2001) or the measles virus receptors (Kitagawa et al., 2005) in their envelope. Gp64 null mutants can be propagated in a helper cell line (providing gp64 expression) and have been used to study structural features of gp64 mutants (Grabherr et al., 2002). These mutants might be advantageous for displaying recombinantly over-expressed membrane proteins on the surface of insect cells.
Aims and methods.
The goal of this project is to use baculovirus surface display to provide relevant protein targets in such a manner that screening assays can be performed (e.g. against yeast-derived Fcab libraries). We will focus on three target proteins: the epidermal growth factor receptor (EGFR), CD64, and CD20. Mutations that lead to EGFR overexpression have been associated with a number of cancers, including lung cancer and glioblastoma multiforme, thus becoming an interesting target for cancer therapy (Kuan et al., 2001). CD64 (cluster of differentiation 64) binds monomeric IgG-type antibodies (Hulett and Hogarth, 1998). The B-cell antigen CD20 constitutes a possible target for treatment of rheumatoid arthritis and is a lymphoma marker (Cragg et al., 2005). In preliminary studies, we have successfully produced soluble forms of EGFR and CD64. However, these soluble variants might have different antigenic properties than their membrane-bound counterparts. Hence, gp64-tagged EGFR, CD64 and CD20 will be expressed in insect cells, and incorporation into the membrane of infected cells as well as into budded virions will be analysed in terms of efficiency, structural properties and glycosylation of these proteins. In addition to the wild-type baculovirus, we are planning to produce gp64-null mutants expressing these membrane proteins. Gp64-null mutants are not infectious in wild-type Sf9 cells, and in case the yield of virus progeny is insufficient, use of a helper cell line providing gp64 is planned to retain viral infectivity. Influenza A virus hemagglutinin and VSV-G (vesicular stomatitis virus coat protein) will serve as positive controls. Further, in vitro translation systems (MembraneMaxTM Protein Expression Kit, Invitrogen) will be tested and compared in terms of feasibility to produce and stabilize the chosen model membrane proteins.
Cragg, M. S., Walshe, C. A., Ivanov, A. O., Glennie, M. J. (2005) The biology of CD20 and its potential as a target for mAb therapy. Curr. Dir. Autoimmun. 8, 140-174
Grabherr, R., Ernst, W. (2002) Baculovirus major envelope protein gp64 and its application in eukaryotic surface display strategies. Recent Res. Dev. Biochem. 3, 445-457
Hulett, M., Hogarth, P. (1998) The second and third extracellular domains of FcgammaRI (CD64) confer the unique high affinity binding of IgG2a. Mol. Immunol. 35, 989-996
Kitagawa, Y., Tani, H., Chang, K. L., Matsunaga, T. M., Moriishi, K., Matsuura, Y. (2005) Ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses. J. Virol. 79, 3639-3652
Kuan, C. T., Wikstrand, C. J., Bigner, D. D. (2001) EGF mutant receptor vIII as a molecular target in cancer therapy. Endocr. Relat. Cancer 8, 83-96
Lai, Y .-K., Lai, C. -W., Liu, H. -J., Hu, Y.-C. (2007) Avian influenza virus hemagglutinin display on baculovirus envelope: Cytoplasmic domain affects virus properties and vaccine potential. Mol. Ther. 15, 989-996
Lu, L., Yu, L., Kwang, J. (2007) Baculovirus surface-displayed hemagglutinin of H5N1 influenza virus sustains its authentic cleavage, hemagglutination activity, and antigenicity . Biochem. Biophys. Res. Commun. 358, 404-409
Mangor, J. T., Monsma, S. A., Johnson, M. C., Blissard, G. W. (2001). A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein. J. Virol. 75, 2544-2556