Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction

Autor: Kazushige Katsura, Takuhiro Ito, Motoaki Wakiyama, Mutsuko Kukimoto-Niino, Yoshikazu Kurosawa, Chie Takemoto, Takayoshi Matsuda, Mikako Shirouzu, Mariko Ikeda, Shigeyuki Yokoyama
Jazyk: angličtina
Rok vydání: 2018
Předmět:
Models
Molecular
0301 basic medicine
Antigen-Antibody Complex
Protein Conformation
Immunoglobulin Variable Region
lcsh:Medicine
Biochemistry
Physical Chemistry
Epitope
Binding Analysis
Epitopes
Protein structure
Post-Translational Modification
Protein disulfide-isomerase
lcsh:Science
Crystallography
Multidisciplinary
biology
Chemistry
Physics
Condensed Matter Physics
Physical Sciences
Crystal Structure
Crystallographic Techniques
Engineering and Technology
Disulfide Bonds
Target protein
Antibody
Research Article
Cell Binding
Cell Physiology
Structural Engineering
Imaging Techniques
X-Ray Crystallography
Structural Characterization
Structural Analysis
Research and Analysis Methods
Immunoglobulin Fab Fragments
03 medical and health sciences
Fluorescence Imaging
Solid State Physics
Humans
Binding site
Chemical Characterization
Chemical Bonding
lcsh:R
Rational design
Biology and Life Sciences
Proteins
Hydrogen Bonding
Cell Biology
030104 developmental biology
biology.protein
lcsh:Q
Binding Sites
Antibody
Zdroj: PLoS ONE, Vol 13, Iss 2, p e0193158 (2018)
PLoS ONE
ISSN: 1932-6203
Popis: Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.
Databáze: OpenAIRE
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