Method for generation of in vivo biotinylated recombinant antibodies by yeast mating.
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.
Scholler
Nathalie
N
Translational Outcomes Research Group, Molecular Diagnostics Program, Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. nscholle@fhcrc.org
Garvik
Barbara
B
Quarles
Travis
T
Jiang
Shaoyi
S
Urban
Nicole
N
D000906:Antibodies / Q000276:immunology*; D000915:Antibody Affinity; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D030501:Epididymal Secretory Proteins / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D020349:Surface Plasmon Resonance; D014162:Transfection; D015003:Yeasts; D023083:beta-Defensins
D000906:Antibodies; C426184:DEFB126 protein, human; D030501:Epididymal Secretory Proteins; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins