Secretion expression of Mt. heat shock protein 70 in Pichia pastoris and identification of the protein.
- Author:
Ming-Li PENG
1
;
Ning LING
;
Hong-Mei XU
;
Yu-Ling QING
;
Hong REN
Author Information
1. Institute for ViraL Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China. minglip55@yahoo.com.cn
- Publication Type:Journal Article
- MeSH:
Animals;
Bacterial Proteins;
genetics;
immunology;
metabolism;
pharmacology;
Blotting, Western;
Cells, Cultured;
Dendritic Cells;
drug effects;
metabolism;
Electrophoresis, Polyacrylamide Gel;
Enzyme-Linked Immunosorbent Assay;
Female;
Genetic Vectors;
genetics;
HSP70 Heat-Shock Proteins;
genetics;
immunology;
metabolism;
pharmacology;
Humans;
Interleukin-12;
metabolism;
Interleukin-6;
metabolism;
Mice;
Mice, Inbred BALB C;
Mycobacterium tuberculosis;
genetics;
metabolism;
Pichia;
genetics;
metabolism;
Rabbits;
Tumor Necrosis Factor-alpha;
metabolism
- From:
Chinese Journal of Biotechnology
2003;19(3):286-290
- CountryChina
- Language:Chinese
-
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.
