We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and Western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K(d)=4.8 x 10(-9) M and K(d)=5.1 x 10(-9) M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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