Immunoglobulin mutant library genetically screened for folding stability exploiting bacterial signal transduction.
A model repertoire of variants of immunoglobulin kappa variable domain REIv with different folding stabilities was generated by oligonucleotide-directed randomization of position 29, a key conserved residue of hypervariable loop 1. Fused to ToxR', the membrane-anchored cytoplasmic domain of the Vibrio cholerae ToxR transcription activator, different members of the library induce different levels of transcription from the ctx promoter in Escherichia coli. Differences in transcription activation correlate positively with folding stabilities of the corresponding REIv domains. Since conformationally stabilized REIv derivatives elicit a dark red colony phenotype on EMB-lactose indicator plates, this procedure constitutes a genetic screen for immunoglobulin folding stability.
Kolmar
H
H
Institut für Molekulare Genetik, Göttingen, F.R.G.
Frisch
C
C
Götze
K
K
Fritz
H J
HJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics; D005803:Genes, Immunoglobulin / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D014734:Vibrio cholerae / Q000737:chemistry*; D001616:beta-Galactosidase / Q000235:genetics; D001618:beta-Lactamases
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria; D002772:Cholera Toxin; D001616:beta-Galactosidase; D001618:beta-Lactamases