Expression,Purification and
Characterization of Regulatory Subunit of
Type I cAMP-dependent Protein Kinase in E.coli
LIU Xu-Lei,LI Hui-Ming,and MA Lan
( National Laboratory of Medical Neurobiology,Shanghai Medical
University,Shanghai 200032,China )
ZHAO Jian,PEI Gang
( 1Shanghai Institute of Cell Biology,the Chinese Academy of
Sciences,Shanghai 200031,China )
Abstract 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.
Key Words PKA；cAMP；expression
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