Secretion expression of Mt. heat shock protein 70 in Pichia pastoris and identification of the protein / 生物工程学报
Chinese Journal of Biotechnology
; (12): 286-290, 2003.
Article
en Zh
| WPRIM
| ID: wpr-270067
Biblioteca responsable:
WPRO
ABSTRACT
To obtain the expression of Mycobacterium tuberculosis heat shock protein 70 in methylotropic yeast. The expression vector pPIC9K-hsp70 was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The result protein was secreted into the supernatant induced by 0.5% methanol at 30 degrees C and purified by centrifugation, ultrafiltration and ATP-agarose. The recombinant Hsp70 was identified by SDS-PAGE, Western blot, mice experiment and effect on the immature DC. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed Hsp70 was about 70 kD and the expressed protein could specifically react with anti-Mt. Hsp70 IgG. And mice immunization indicated the expressed hsp70 had immunogenicity. Hsp70 could induce DC maturation and release Th1 cytokine. The secreted 70 kD protein was about 120 mg/L which accounted for more than 30% of the total supernatant protein and was purified to electrophoretic purity. The Hsp70, which had the biological activity, is successfully secretorily expressed in the Pichia pastoris GS115.
Texto completo:
1
Base de datos:
WPRIM
Asunto principal:
Farmacología
/
Pichia
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Proteínas Bacterianas
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Células Dendríticas
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Ensayo de Inmunoadsorción Enzimática
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Células Cultivadas
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Western Blotting
/
Interleucina-6
/
Factor de Necrosis Tumoral alfa
/
Interleucina-12
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Animals
/
Female
/
Humans
Idioma:
Zh
Revista:
Chinese Journal of Biotechnology
Año:
2003
Tipo del documento:
Article