title: Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries.
abstract: New phage display vectors for in vivo recombination of immunoglobulin (Ig) heavy (VH) and light (VL) chain variable genes, to make single-chain Fv fragments (scFv), were constructed. The VH and VL genes of monoclonal antibody (mAb) EP-5C7, which binds to both human E- and P-selectin, were cloned into a pUC19-derived plasmid vector, pCW93, and a pACYC184-derived phagemid vector, pCW99, respectively. Upon induction of Cre recombinase (phage P1 recombinase), the VH and VL genes were efficiently recombined into the same plasmid via the two loxP sites (phage P1 recombination sites), one located downstream from a VH gene in pCW93 and another upstream from a VL gene in pCW99. In the resulting phagemid, the loxP sequence also encodes a polypeptide linker connecting the VH and VL domains to form a scFv of EP-5C7. Whether expressed on the phage surface or as a soluble form, the EP-5C7 scFv showed specific binding to human E- and P-selectin. This phagemid vector system provides a way to recombine VH and VL gene libraries efficiently in vivo to make extremely large Ig combinatorial libraries.
authors.affiliation: Protein Design Labs, Inc., Mountain View, CA 94043, USA. naoya@pdl.com
authors.forename: N
authors.initials: N
authors.lastname: Tsurushita
authors.forename: H
authors.initials: H
authors.lastname: Fu
authors.forename: C
authors.initials: C
authors.lastname: Warren
mesh_terms: D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D017931:DNA Primers; D019040:E-Selectin / Q000378:metabolism; D005822:Genetic Vectors; D015698:Genomic Library; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics* / Q000378:metabolism; D019426:Integrases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019007:P-Selectin / Q000378:metabolism; D010957:Plasmids; D011995:Recombination, Genetic; D014764:Viral Proteins
chemical_list: D017931:DNA Primers; D019040:E-Selectin; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019007:P-Selectin; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases