Expression, Purification and Characterization of Regulatory Subunit of Type I cAMP-dependent Protein Kinase in E.coli.
Regulatory subunit of type I cAMP-dependent protein kinase (RI) possesses two cAMP-binding sites with high affinity for cAMP. In the current study, human RI cDNA fragment coding for residue 93-381 was cloned from neuronal cells. After subcloning into pET30a, the recombinant RI was expressed in E.coli BL21. Considerable amount of soluble RI subunit was produced in BL21 upon induction of IPTG. RI was purified to homogeneity over 95% after His-bind metal chelation chromatography. Approximately 8 mg pure RI was obtained from 0.1 liter of BL21 culture. Human RI prepared with this method binds to cAMP specifically and therefore can be used in cAMP assay.
National Laboratory of Medical Neurobiology, Shanghai Medical University, Shanghai 200032, China. lanma@shmu.edu.cn
Xu-Lei
XL
Liu
Hui-Ming
HM
Li
Jian
J
Zhao
Gang
G
Pei
Lan
L
Ma