An antibody VH domain with a lox-Cre site integrated into its coding region: bacterial recombination within a single polypeptide chain.
Bacterial lox-Cre recombination within a single antibody VH domain was achieved through integration of a loxP site into its coding sequence. The 5' half of the VH gene, in which the H2 loop was replaced by a mutant loxP site, was fused to geneIII in an 'acceptor' fd-phage vector containing also a wild type loxP site. With a 'donor' plasmid vector harbouring the 3' half of the VH gene flanked by the same, differing loxP sites it recombined into a full-length VH with the loxP site-H2 loop. This VH was purified from bacterial periplasm, where it folded into a typical immunoglobulin domain. The system allows the generation of large VH repertoires using lox-Cre recombination.
MRC Laboratory of Molecular Biology, Cambridge, UK.
J
J
Davies
L
L
Riechmann
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005822:Genetic Vectors; D006358:Hot Temperature; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D017912:Inovirus / Q000235:genetics; D019426:Integrases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D013816:Thermodynamics; D014764:Viral Proteins
D007135:Immunoglobulin Variable Region; D014764:Viral Proteins; D004247:DNA; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases