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


CONCLUSIONS
We have successfully purified and isolated PKR interacting proteins,  thus  providing a basis for future research.
Department of Immunology, Sun Yat-sen University, Guangzhou, China. xiejiong59@163.com
Jiong
J
Xie
Jun
J
Xia
Pei-fen
PF
Zhang
Yu-ye
YY
Li
Min-hao
MH
Wu
Xi
X
Huang
Ping
P
Zhang
D003001:Cloning, Molecular; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D019892:eIF-2 Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D019892:eIF-2 Kinase