Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones.
Nat Biotechnol
; 15(1): 79-84, 1997 Jan.
Article
em En
| MEDLINE
| ID: mdl-9035111
ABSTRACT
To create a rapid system to test the effect of sequence changes on recombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translation system. Functional antibodies with antigen-binding activity are obtained only if disulfide formation and rearrangement is allowed to take place during the translation reaction. The inclusion of protein disulfide isomerase (PDI) leads to a threefold increase in yield over that obtained in the presence of glutathione redox systems. DsbA had no such effect, indicating that disulfide shuffing, and not net formation, is the crucial yield-limiting step. The addition of the molecular chaperones DnaK and DnaJ increased the amount of soluble protein but not the amount of functional scFv, which appears to be limited entirely by correct disulfide formation. None of these factors significantly influenced total protein synthesis. In the presence of PDI, chaperones, reduced glutathione and oxidized glutathione, 50% of the scFv produced (about 8 micrograms/ml in only 15 min) could be recovered from immobilized antigen.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Biossíntese de Proteínas
/
Chaperonas Moleculares
/
Isomerases
/
Formação de Anticorpos
Idioma:
En
Revista:
Nat Biotechnol
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Suíça