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

Soo Jeong Lee, Ji Hong Bong, Jaeyong Jung, Jeong Soo Sung, Min Jung Kang, Joachim Jose, Jae Chul Pyun

Research output: Contribution to journalArticlepeer-review

Abstract

This study aimed to isolate FV-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 FV-antibodies on the outer membrane of E. coli using autodisplay technology, (2) screening the FV-antibodies with biotin-binding activity from the FV-antibody library, and (3) synthesizing peptides (molecular weight of several kDa) from the biotin-binding amino acid sequence of FV-antibodies. An FV-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 FV-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 (KD) of Fv-antibodies against biotin was calculated and the binding properties of the screened FV-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 FV-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.

Original languageEnglish
Article number338627
JournalAnalytica Chimica Acta
Volume1169
DOIs
Publication statusPublished - 2021 Jul 18

Bibliographical note

Funding Information:
This work is supported by the National Research Foundation of Korea [grant numbers: NRF-2020R1A2B5B01002187 and NRF-2020R1A5A101913111 ].

Publisher Copyright:
© 2021 Elsevier B.V.

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy

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