[Expression and purification of recombinant hypodermin C in Pichia pastoris].
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.
Gao
Xing-Chun
XC
Shanghai Institute of Animal Parasitology, Chinese Academy of Agricultural Sciences, Shanghai 200232, China.
Xu
Mei-Qian
MQ
He
Guo-Sheng
GS
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005780:Gelatin / Q000378:metabolism; D015870:Gene Expression; D007106:Immune Sera / Q000276:immunology; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity; D014170:Transformation, Genetic
D007106:Immune Sera; D011994:Recombinant Proteins; D005780:Gelatin; D012697:Serine Endopeptidases; C070019:hypodermin C