C-terminal His-tag Fusion Expression and Purification of Truncated cAMP-dependent Protein Kinase.
The truncated mCalpha lacking 3'-coding sequence of 96 base paires was fused with a His-tag (mCalpha4H) and a C-terminal fusion expression plasmid pZP mCalpha4H was constructed. With the induction of IPTG, the Expressed mCalpha4H was up to about 20% of the total bacterial proteins in E. coli BL21 (DE3). Using immobilized metal (Ni(2+)) chelation affinity chromatography, the target protein mCalpha4H was purified from crude lysates and inclusion bodies respectively. The results of in vitro and in vivo myristoylation assay showed that the purifed mCalpha4H is a substrate of NMT as the mCalpha.
Xu
Zheng-Ping
ZP
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China.
Duan
Zhi-Jun
ZJ
Chen
Chang-Zheng
CZ
Li
Bo-Liang
BL