Lee, Soo Jeong Bong, Ji-Hong Jung, Jaeyong Sung, Jeong Soo Kang, Min-Jung Jose, Joachim Pyun, Jae-Chul 2024-01-19T14:03:49Z 2024-01-19T14:03:49Z 2021-09-05 2021-07-18 0003-2670 https://pubs.kist.re.kr/handle/201004/116686 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. English ELSEVIER SITE-DIRECTED MUTAGENESIS ESCHERICHIA-COLI PEPTIDE LIBRARY AVIDIN IMMUNOASSAY PROTEIN CELLS DOMAINS CDR3 RECOGNITION Screening of biotin-binding F-V-antibodies from autodisplayed F-V-library on E. coli outer membrane Article 10.1016/j.aca.2021.338627 1 ANALYTICA CHIMICA ACTA, v.1169 ANALYTICA CHIMICA ACTA 1169 scie scopus 000653032800002 2-s2.0-85106215910 Chemistry, Analytical Chemistry Article SITE-DIRECTED MUTAGENESIS ESCHERICHIA-COLI PEPTIDE LIBRARY AVIDIN IMMUNOASSAY PROTEIN CELLS DOMAINS CDR3 RECOGNITION F-V-Antibody Antibody library Autodisplay Biotin Z-domain