Expression and protein chemistry yielding crystallization of the catalytic domain of ADAM17 complexed with a hydroxamate inhibitor.
Protein Expr Purif
; 52(2): 313-9, 2007 Apr.
Article
em En
| MEDLINE
| ID: mdl-17169570
ABSTRACT
The membrane-anchored metalloproteinase ADAM17 (TNF-alpha converting enzyme; TACE; EC 3.4.24.86) continues to be an attractive drug target in inflammatory diseases and cancer. Cocrystallization of its catalytic domain with a lead compound was complicated by the tenacious retention of the prodomain that has been shown to be enhanced if ADAM17 is expressed without the disintegrin/cysteine-rich domain that normally follows the N-terminal metalloproteinase. When a truncated form of ADAM17 composed of the signal peptide with the pro- and catalytic domains was expressed in baculovirus-infected insect cells, the major secreted product was a ternary complex of two prodomain fragments with the catalytic domain. The component polypeptides of the ternary complex were characterized by N-terminal analysis and mass spectrometry. Internal cleavage of the propeptide occurred following Arg-58, and a carboxypeptidase variably removed up to three basic residues from the newly created C-terminus. Cleavage at the C-terminus of the propeptide occurred after Arg-214. To prepare ADAM17 for crystal growth, a drug-like inhibitor was used to displace the propeptide and the complex of the catalytic domain with the inhibitor was isolated by size-exclusion chromatography and crystallized.
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Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Domínio Catalítico
/
Proteínas ADAM
/
Ácidos Hidroxâmicos
Limite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2007
Tipo de documento:
Article
País de afiliação:
Estados Unidos