Overexpression and refolding of thioredoxin/TRAIL fusion from inclusion bodies and further purification of TRAIL after cleavage by enteropeptidase.
Biotechnol Lett
; 29(10): 1567-73, 2007 Oct.
Article
em En
| MEDLINE
| ID: mdl-17609857
The human TRAIL gene (encoding residues 114-281) was synthesized by PCR and cloned into plasmid pET-32a. High level expression (1.5 g l(-1)) of thioredoxin/TRAIL fusion was achieved in Escherichia coli strain BL21(DE3), mainly as inclusion bodies. Refolded fusion thioredoxin/TRAIL was cleaved by enteropeptidase and TRAIL was separated from thioredoxin on Ni-NTA agarose. High yield (400 mg l(-1)) of TRAIL without N-terminal methionine and His tag was obtained. Sedimentation coefficient demonstrated that 98% of TRAIL formed trimers. TRAIL formed crystals of space group P3 (1) with unit-cell dimensions a = b = 72.5 A, c = 141.5 A. Apoptosis induced in HeLa cells by purified TRAIL was 5-fold enhanced by emetine.
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Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Tiorredoxinas
/
Proteínas Recombinantes de Fusão
/
Enteropeptidase
/
Corpos de Inclusão
/
Ligante Indutor de Apoptose Relacionado a TNF
Limite:
Humans
Idioma:
En
Revista:
Biotechnol Lett
Ano de publicação:
2007
Tipo de documento:
Article