Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework.
Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.
der Maur
Adrian Auf
AA
ESBATech AG, Zürich-Schlieren, Switzerland.
Zahnd
Christian
C
Fischer
Franziska
F
Spinelli
Silvia
S
Honegger
Annemarie
A
Cambillau
Christian
C
Escher
Dominik
D
Plückthun
Andreas
A
Barberis
Alcide
A
D000595:Amino Acid Sequence; D000941:Antigens / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D005803:Genes, Immunoglobulin; D017930:Genes, Reporter; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D017510:Protein Folding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000941:Antigens; D004268:DNA-Binding Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D011494:Protein Kinases