Establishment and application of a tandem affinity purification system of innate immune regulatory protein PKR
10.3760/cma.j.issn.0254-5101.2011.06.002
- VernacularTitle:固有免疫调控蛋白PKR串联亲和纯化系统的建立及应用
- Author:
Yuye LI
;
Zhaoduan LIANG
;
Siyu WU
;
Jiong XIE
;
Junfang HE
;
Minhao WU
;
Xi HUANG
;
Ping ZHANG
- Publication Type:Journal Article
- Keywords:
Double-stranded RNA;
Protein kinase PKR;
Affinity;
Purification
- From:
Chinese Journal of Microbiology and Immunology
2011;31(6):487-491
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a tandem affinity purification(TAP) system of innate immune-regulatory protein PKR and analyze PKR function, for the future screen and identification of novel PKR-interaction proteins. Methods PKR gene was amplified by PCR, and then cloned into a mammalian expression vector pcTAP-A. Recombinant pcTAP-PKR was transfected into PKR knock-down(PKRkd) HeLa cells by LipofectAMINE 2000,and the PKR overexpressed HeLa cells were harvested for mitogen-activated protein kinases(MAPK) activation analysis. Cell extracts of PKR overexpressed cells were purified using TAP kit and examined by Western blot. Results Cal modulin resin(CBP) and streptavidin resin(SBP) tagged PKR was detected in PKRkd HeLa cells as early as 24 h upon transfection with pcTAP-PKR, and its expression decreased at later time points. The overexpression of PKR was autophosphorylated, and thus involved in the regulation of MAPK actviation. After small-scale TAP kit purification, PKR protein was detectable by Western blot. Conclusion We have successfully established a TAP system that over-expresses functional PKR, providing a useful tool for the future study on the identification of PKR interacting proteins.
