Screening of biotin-binding F_V-antibodies from autodisplayed F_V-library on E. coli outer membrane

This study aimed to isolate F_V-antibodies with biotin-binding activity from a F_V-antibody library that was successfully screened on the outer membrane of E. coli. The aims were achieved by (1) preparing a library of F_V-antibodies on the outer membrane of E. coli using autodisplay technology, (2) screening the F_V-antibodies with biotin-binding activity from the F_V-antibody library, and (3) synthesizing peptides (molecular weight of several kDa) from the biotin-binding amino acid sequence of F_V-antibodies. An F_V-antibody library with a diversity of 1.7 × 10⁵ clones was prepared on the outer membrane of E. coli, using a surface display method called autodisplay technology. For the screening of biotin-binding F_V-antibodies, the fluorescence-labeled biotin was introduced into the library, and the target E. coli with biotin-binding activity were screened using flow cytometry. For the screened E. coli clones, the binding affinity (K_D) of Fv-antibodies against biotin was calculated and the binding properties of the screened F_V-antibody were analyzed through competition assay with a synthetic peptide having the biotin-like activity. From the FRET experiment with the synthetic peptide corresponding to the CDR3 region of the screened Fv-antibody, the biotin-binding activity of the screened F_V-antibody was proved to be originated from the CDR3. Finally, the applicability of the biotin-binding domain was demonstrated through the co-expression with a protein called Z-domain with antibody binding activity.