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 hemagglutinin and expressed on the surface of baculovirus infected insect cells. In order to improve the antigenicity of the epitope within the hemagglutinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope. This pool of hemagglutinin genes was directly cloned into the baculovirus Ac-omega. To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capacity of individual viral clones. Using fluorescence activated cell sorting (FACS) we isolated a baculovirus clone displaying the epitope with markedly increased binding capacity out of a pool of 8000 variants in only one sorting step. Binding properties of the identified ligand were examined by FACS performing a competition assay.
Ernst
W
W
Insitute of Applied Microbiology, University of Agriculture, Muthgasse 18, A-1190 Vienna, Austria.
Grabherr
R
R
Wegner
D
D
Borth
N
N
Grassauer
A
A
Katinger
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D000939:Epitopes / Q000096:biosynthesis* / Q000737:chemistry; D005434:Flow Cytometry; D015700:HIV Envelope Protein gp41 / Q000096:biosynthesis / Q000737:chemistry; D015497:HIV-1 / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D018411:Spodoptera; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015700:HIV Envelope Protein gp41; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D019151:Peptide Library; D001616:beta-Galactosidase