Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.
Protein Sci
; 4(10): 2149-55, 1995 Oct.
Article
em En
| MEDLINE
| ID: mdl-8535252
ABSTRACT
Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
/
Cisteína Endopeptidases
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Protein Sci
Assunto da revista:
BIOQUIMICA
Ano de publicação:
1995
Tipo de documento:
Article
País de afiliação:
Estados Unidos