Expression of recombinant human A20 in Pichia pastoris GS115
- VernacularTitle:重组人A20蛋白在巴斯德毕赤酵母GS115中的表达
- Author:
Lijuan WU
- Publication Type:Journal Article
- Keywords:
A20;
gene expression;
Pichia pastoris;
genetic engineering
- From:Journal of Third Military Medical University
1983;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective Human A20 is one of the endogenetic regulating proteins of inflammation in vivo and play important roles in preventing uncontrolled inflammatory response as well as endogenetic cellular protection.Our aim is to prepare a recombinant protein of human A20(rhA20) in Pichia pastoris and then research its roles in sepsis therapy.Methods rhA20 yeast expression vector yevCFP-hA20 was constructed by genetic engineering.Pichia pastoris with linearized yevCFP-hA20 was electroporated.The high copy clones were screened with MD/His~(-) medium and YPD/G418 gradient medium.The number of hA20 in yeast genome was detected by quantitative PCR.The rhA20 was expressed by methanol induction and identified by SDS-PAGE and Western blotting.The optimum conditions(including pH and ingredient of induced medium as well as the temperature of induced culture) were investigated.Results The molecular weight of rhA20 is about 93?10~3.After expression condition's optimization by decreasing the pH of BMMY medium,adding a low dosage of EDTA and the substrate of protease,as well as decreasing the induced temperature,the full expressing products was up to 11 times,rose to 18.24% in total protein and achieved to 254.1 024.52?g/ml.Conclusion we have successfully expressed rhA20 in Pichia pastoris GS115.The expression amount of full rhA20 increases clearly by progressing the ingredient and pH of BMMY and using a lower inducing temperature.This starts the biosynthesis of hA20 in vitro.A new solving regime is provided to other biologist when they meet product's degradation by using Pichia pastoris GS115.
