[Cloning and expression of PKR gene and affinity purification of PKR interacting proteins].

AIM:To clone and express protein kinase regulated by double-stranded RNA (PKR) gene, and to purify and isolate PKR interacting proteins. METHODS:By using specific primers, PKR gene fragments tagged with HA and FLAG (FLAG-PKR-HA, and HA-PKR-FLAG) were amplified by PCR, and cloned into pSG5 vector. The recombinant plasmids were transfected into PKR knockdown (PKR(kd);) HeLa cells by Lipfectamine(TM); 2000. The expression of tagged PKR was confirmed by Western blotting. Finally, the PKR interacting proteins were isolated by tandem affinity purification (TAP) system using HA and FLAG antibodies, and visualized by Western blotting and SDS-PAGE silver staining. RESULTS:PKR expression plasmids were constructed and TAP system was successfully established. Silver staining showed that PKR, and two potential PKR interacting protein fragments were isolated by SDS-PAGE. CONCLUSION:We have successfully purified and isolated PKR interacting proteins, thus providing a basis for future research.