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

Abstract

This study aimed to isolate F-V-antibodies with biotin-binding activity from a FV-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 FVantibodies 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 FVantibody library with a diversity of 1.7 x 10(5) 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 biotinbinding activity were screened using flow cytometry. For the screened E. coli clones, the binding affinity (K-D) of F-v-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. (C) 2021 Elsevier B.V. All rights reserved.