Expression, refolding and purification of a human interleukin-17A variant.
Cytokine
; 53(1): 107-14, 2011 Jan.
Article
em En
| MEDLINE
| ID: mdl-20674388
ABSTRACT
A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Interleucina-17
/
Proteínas Mutantes
/
Redobramento de Proteína
Limite:
Humans
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article