In vitro folding and thermodynamic stability of an antibody fragment selected in vivo for high expression levels in Escherichia coli cytoplasm.
J Mol Biol
; 292(4): 921-9, 1999 Oct 01.
Article
em En
| MEDLINE
| ID: mdl-10525415
ABSTRACT
We recently isolated a mutant of a human anti-beta-galactosidase single chain antibody fragment (scFv) able to fold at high levels in Escherichia coli cytoplasm. When targeted to the periplasm, this mutant and the wild-type scFv are both expressed at comparable levels in a soluble, active and oxidized form. If a reducing agent is added to the growth medium, only the mutant scFv is still able to fold, showing that in vivo aggregation is a direct consequence of the lack of disulphide bond formation and not of the cellular localization. In vitro denaturation/renaturation experiments show that the mutant protein is more stable than the wild-type scFv. Furthermore, refolding kinetics under reducing conditions show that the mutant folds faster than the wild-type protein. Aggregation does not proceed from the native or unfolded conformation of the protein, but from a species only present during the unfolding/refolding transition. In conclusion, the in vivo properties of the mutant scFv can be explained by, first, an increase in the stability of the protein in order to tolerate the removal of the two disulphide bonds and, second, a modification of its folding properties that reduces the kinetic competition between folding and aggregation of a reduced folding intermediate.
Buscar no Google
Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Fragmentos de Imunoglobulinas
/
Dobramento de Proteína
/
Citoplasma
/
Escherichia coli
Limite:
Humans
Idioma:
En
Revista:
J Mol Biol
Ano de publicação:
1999
Tipo de documento:
Article