[Secretion expression of Mt. heat shock protein 70 in Pichia pastoris and identification of the protein].
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.
Peng
Ming-Li
ML
Institute for ViraL Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China. minglip55@yahoo.com.cn
Ling
Ning
N
Xu
Hong-Mei
HM
Qing
Yu-Ling
YL
Ren
Hong
H
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D018664:Interleukin-12 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12