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Protein Eng Des Sel. 2013 Oct;26(10):655-62. doi: 10.1093/protein/gzt050. Epub 2013 Sep 24.

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

Author information

  • 1Department of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.

Abstract

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

KEYWORDS:

intron; library; screening; signal sequence; splicing

PMID:
24065833
[PubMed - indexed for MEDLINE]
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