摘要
目的建立固定金属亲和层析法(Immobilized metal affinity chromatography,IMAC)纯化重组Kringle 5蛋白的工艺,为Kringle 5蛋白的制备及生物学活性研究奠定基础。方法常规制备IMAC蛋白上样样品,分别采用pH梯度洗脱和咪唑梯度洗脱的方法获取Kringle 5蛋白洗脱液,分段收集洗脱样品,SDS-PAGE检测蛋白纯度,Western blot鉴定其反应原性。结果咪唑梯度洗脱法获得的Kringle 5蛋白纯度达98%以上,得率为21.3 mg/柱体积,但不能充分将Kringle 5蛋白与杂蛋白分离;pH梯度洗脱法获得的Kringle 5蛋白纯度在98%以上,得率为62.7 mg/柱体积,且可与杂蛋白充分分离。结论 已建立了重组Kringle 5蛋白的纯化方法,为Kringle 5蛋白的制备及生物学活性研究奠定了基础。
Objective To develop a purification procedure for recombinant Kringle 5 protein by immobilized metal affinity chromatography（IMAC） and lay a foundation of preparation and study on bioactivity of the protein.Methods Protein samples were prepared by routine method and loaded onto IMAC column,then eluted by pH gradient elution and imidazole gradient elution respectively.Various fractions of eluate were collected,then identified for protein purity by SDS-PAGE and for reactogenicity by Western blot.Results The Kringle 5 protein obtained by imidazole gradient elution reached a purity of more than 98%,of which the yield was 21.3 mg / column volume.However,Kringle 5 protein could not be separated from foreign proteins completely.The Kringle 5 protein obtained by pH gradient elution reached a purity of more than 98% and was completely separated from foreign protein,of which the yield was 62.7 mg / column volume.Conclusion A method for purification of Kringle 5 protein was developed,which laid a foundation of preparation and study on bioactivity of the protein.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第9期1101-1104,共4页
Chinese Journal of Biologicals
基金
山西省科技攻关项目(20080321078)
山西医科大学博士启动基金(03200823)
关键词
KRINGLE
5蛋白
固定金属亲和层析
纯化
Kringle 5 protein
Immobilized metal affinity chromatography（IMAC）
Purification
