On-off switching of enzymatic reaction by recombinant calmodulin on a solid-phase matrix.
A fusion protein consisting of human calmodulin (CaM) and glutathione S-transferase (GST) was produced by gene fusion. The fusion protein was overexpressed in Escherichia coli as a soluble form and purified with one-step affinity chromatography using glutathione-Sepharose. The protein had the modulating activity of CaM and the binding capability to glutathione of GST. Phosphodiesterase, which is a CaM dependent enzyme, was activated by the fusion protein, with the Ca2+ level equal to the level equivalent to a native CaM. Furthermore, CaM could be immobilized on a solid-phase matrix through the use of GST moiety while its modulating activity was retained. Phosphodiesterase activity was switched on and off by the immobilized CaM with or without Ca2+, and repeated use of CaM was demonstrated.
Kobatake
E
E
Department of Bioengineering, Tokyo Institute of Technology, Yokohama, Japan.
Mitomo
K
K
Haruyama
T
T
Aizawa
M
M
Deng
G Y
GY
Kato
S
S
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000378:metabolism; D004926:Escherichia coli; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D002147:Calmodulin; D017931:DNA Primers; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D005978:Glutathione; D002118:Calcium