Expression, refolding, and characterization of recombinant thrombopoietin/stem cell factor fusion protein in Escherichia coli.
Appl Microbiol Biotechnol
; 74(4): 836-42, 2007 Mar.
Article
en En
| MEDLINE
| ID: mdl-17123074
Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx) fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method. Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni(2+) affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Trombopoyetina
/
Proteínas Recombinantes de Fusión
/
Factor de Células Madre
Límite:
Humans
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Año:
2007
Tipo del documento:
Article
Pais de publicación:
Alemania