Enzymatic deglutathionylation to generate interleukin-4 cysteine muteins with free thiol.
Bioconjug Chem
; 23(7): 1396-405, 2012 Jul 18.
Article
em En
| MEDLINE
| ID: mdl-22681442
ABSTRACT
Interleukin-4 (IL-4) is a prototypical regulator protein of the immune system that is crucial for the pathogenesis and maintenance of asthma and other atopic diseases. It, together with IL-13, uses the IL-4 receptor α chain (IL-4Rα) to signal into immune and other cells. An IL-4 mutein acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. Here, it is described how IL-4 muteins containing a single engineered cysteine with a free thiol can be prepared and used for site-specific chemical modification. The muteins were initially expressed in E. coli, refolded, and purified, but not in a fully reduced nonconjugated form. Attempts to reduce the cysteine chemically failed because the native disulfide bonds of IL-4 were also reduced under similar conditions. Therefore, an enzymatic procedure was developed to reduce glutathionylated IL-4 cysteine muteins employing glutaredoxin and reduced glutathione. Cysteine muteins engineered at four different positions around the IL-4Rα binding site were enzymatically reduced at different rates. All muteins were prepared with free thiol in reasonable yield and were modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effect on IL-4Rα binding of cysteine substitution and of the site-specific modification by glutathione, N-ethylmaleimide (NEM), or a branched 2.36 kDa poly(ethylene glycol) (PEG) will be discussed.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Compostos de Sulfidrila
/
Engenharia de Proteínas
/
Interleucina-4
/
Cisteína
/
Glutationa
/
Glutationa Redutase
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Bioconjug Chem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Alemanha