The Construction and Functional Study of Protein Kinase Inhibitor Phage.
A DNA fragment, which encoded the heat-stable protein kinase inhibitor (PKI) (5-24) of camp-dependent protein kinase (cAPK), was synthesized and cloned into phage display vector fe-tet-DOG1. Thus, PKI(5-24) was displayed on the surface of phage fd, which was termed PKI phage (cAPK inhibitor phage), in a form fused with gene III protein (g3p). It was showed that PKI phage not only repressed cAPK effectively, but also bound with the immobilized recombinant His(6)-tag mouse cAPK-Calpha( His(6)-mCalpha) specifically. The bound PKI phages could be quantitatively eluted under acidic conditions. Model affinity screening demonstrated that PKI phages could be selectively enriched from the mixture of PKI phage and wild-type phages (1:10(8)) using affinity chromatography of immobilized His(6)-mCalpha. These results suggest that selecting protein kinase inhibitor by phage display technique is feasible.
Chen
Chang-Zheng
CZ
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China.
Yang
Xin-Ying
XY
Xia
Qi-Chang
QC
Li
Bo-Liang
BL
Wang
Ying-Lai
YL