Single-chain Fv antibody covalently linked to antigen peptides and its structural evaluation.
Anal Biochem
; 629: 114312, 2021 09 15.
Article
em En
| MEDLINE
| ID: mdl-34302799
ABSTRACT
The monoclonal antibody G2 specifically recognizes different peptides. The single-chain Fv (scFv) antibodies of G2 covalently linked to antigen peptides, Pep18mer and Pep395, via a flexible linker were expressed in Escherichia coli in the insoluble fraction, and were solubilized using guanidine HCl, followed by refolding. We analyzed the folding thermodynamics of the refolded proteins, purified as monomers using size-exclusion chromatography (SEC). The results of the differential scanning calorimetry (DSC) showed that the thermal stabilities of antigen peptide-linked G2 scFvs were higher than those of antigen-free G2 scFv in the absence or presence of antigen peptides. The folding thermodynamics further indicated how the antigen-antibody affinity affect the intramolecular interactions. The combination of SEC and DSC experiments could confirm the folding correctness of antigen peptide-linked G2 scFvs and could be applied for "structural screening" of refolded proteins in the case that the "functional screening" like antigen binding is difficult to apply. The present method to covalently link the peptide would contribute to the stable complex structure, and would be widely applied to other antibodies recognizing peptide antigens.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
/
Anticorpos de Cadeia Única
/
Antígenos
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article