Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1
Braz. j. med. biol. res
; 47(7): 540-547, 07/2014. tab, graf
Article
em En
| LILACS
| ID: lil-712968
Biblioteca responsável:
BR1.1
ABSTRACT
Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.
Palavras-chave
Texto completo:
1
Índice:
LILACS
Assunto principal:
Fragmentos de Imunoglobulinas
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Expressão Gênica
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Molécula 1 de Adesão Intercelular
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Renaturação Proteica
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Anticorpos de Cadeia Única
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Redobramento de Proteína
Limite:
Animals
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Female
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Humans
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Male
Idioma:
En
Revista:
Braz. j. med. biol. res
Assunto da revista:
BIOLOGIA
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MEDICINA
Ano de publicação:
2014
Tipo de documento:
Article
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Project document