Expression and purification of recombinant hypodermin C in Pichia pastoris.
- Author:
Xing-Chun GAO
1
;
Mei-Qian XU
;
Guo-Sheng HE
Author Information
1. Shanghai Institute of Animal Parasitology, Chinese Academy of Agricultural Sciences, Shanghai 200232, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Blotting, Western;
Cell Line;
Chromatography, Ion Exchange;
Cloning, Molecular;
Electrophoresis, Polyacrylamide Gel;
Gelatin;
metabolism;
Gene Expression;
Immune Sera;
immunology;
Pichia;
genetics;
Plasmids;
genetics;
Rabbits;
Recombinant Proteins;
isolation & purification;
metabolism;
Serine Endopeptidases;
genetics;
immunology;
metabolism;
Substrate Specificity;
Transformation, Genetic
- From:
Chinese Journal of Biotechnology
2007;23(3):552-556
- CountryChina
- Language:Chinese
-
Abstract:
Hypodermin C (HC) cDNA was amplified from recombinant pGEM - T/HC, cloned in frame with the signal sequence in yeast vector pPIC9k. The plasmid was linerarized and transformed into Pichia pastoris GS115 strain by electroporation method. Recombinant strain was screened by G418 resistant, and further confirmed by PCR. The recombinant strain which contains insert was induced in the medium containing 0.5% methanol. The supernatant was collected and then purified by anion exchange chromatography. SDS-PAGE indicated that the target protein is around 28kD. Western-blot showed it can react with rabbit-anti HC serum. Gelatin substrate SDS-PAGE displayed it had enzyme activity. Provided a method to produce enough antigens for carrying out extensive immunological analyses.
