Efficient production of antibodies against a mammalian integral membrane protein by phage display

2011 
The application of phage display technology to mamma-lian proteins with multiple transmembrane regions hashad limited success due to the difficulty in generatingthese proteins in sufficient amounts and purity. Wereport here a method that can be easily and generallyapplied to sorting of phage display libraries with multi-span protein targets solubilized in detergent. A keyfeature of this approach is the production of biotinylatedmultispan proteins in virions of a baculovirus vector thatallows library panning without prior purification of thetarget protein. We obtained Fab fragments from a nai¨vesynthetic antibody phage library that, when engineeredinto full-length immunoglobulin (Ig)G, specifically bindcells expressing claudin-1, a protein with four transmem-brane regions that is used as an entry co-receptor by thehepatitis C virus (HCV). Affinity-matured variants of oneof these antibodies efficiently inhibited HCV infection.The use of baculovirus particles as a source of mamma-lian multispan protein facilitates the application of phagedisplay to this difficult class of proteins.Keywords: phage display/membrane protein/antibody/claudin/baculovirusIntroductionPhage display is widely used to obtain ligands for appli-cations ranging from structural biology to therapeutic useand is particularly well-suited when there is a need for high-throughput or the ability to obtain non-antibody ligands.However, phage display has had limited success in obtainingligands to mammalian proteins with multiple transmembraneregions, a major class of current and potential drug targets(Hopkins and Groom, 2002; Russ and Lampel, 2005). Thetwo main approaches that have been used for application ofphage display to membrane proteins are cell-based selectionand selection with purified membrane proteins either in sol-ution or in reconstituted lipid bilayers. Selection of bindersto multispan membrane protein targets overexpressed onmammalian cells often requires complex steps of negativeselection to avoid the unwanted specificities that are usuallydominant (Sawyer et al., 1997; Giordano et al., 2001;Souriau et al., 2004). The use of purified mammalian multi-span protein for library sorting is more effective at obtainingantibodies from phage display libraries than panning onwhole cells but it is usually hampered by the need for time-consuming optimization of conditions for expression, purifi-cation, and sometimes reconstitution of these proteins inlipid bilayers (Jespersen et al., 2000; Mirzabekov et al.,2000; Milovnik et al., 2009). As a consequence, reports ofapplication of phage display with solubilized multispan pro-teins are limited to those that have been optimized forexpression and stability for structural studies (Milovniket al., 2009; Uysal et al., 2009). A rapid and simple methodenabling phage selection against multispan transmembraneproteins would facilitate the identification of novel antibodyfragments for structural studies or therapeutic development.The selection of phage display libraries with a variety ofmultispan proteins that have not been extensively character-ized would ideally require limited amounts of protein andlittle if any protein purification to avoid protein loss and stab-ility issues. The baculovirus expression system is widelyused for the expression of mammalian membrane proteinsand requires little time from cloning the gene of interest inexpression vectors to protein expression. Membrane proteinsare traditionally harvested with cells, often requiring purifi-cation of the active recombinant multispan protein in severalsteps. However, a significant portion of the expressed mem-brane proteins is also incorporated into virions from thebaculovirus vector during budding at the cell surface, wherethe virus acquires its envelope membrane from the host cellmembrane (Loisel et al., 1997; Masuda et al., 2003; Hayashiet al., 2004). We reasoned that incorporation of membraneproteins by virions could be used to enrich membrane pro-teins to levels sufficient for sorting of phage display librarieswithout the need for protein purification for three mainreasons. First, a major fraction of contaminants is associatedwith the capsids of viral particles that can be easily andrapidly removed from detergent-solubilized samples by asimple centrifugation step. Second, virions could be used toseparate membrane proteins biotinylated in vivo from otherphysiologically biotinylated proteins without the need forpurification of membrane and organelle fractions, allowingimmediate use of protein without prior chemical or enzy-matic biotinylation and free biotin removal steps. Finally,
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