Calmodulin (CaM), a calcium binding protein, regulates important biochemical processes in signal transduction and gene expression. Particularly, we are investigating the roles of CaM in nitric oxide synthase, superoxides produced by NADPH oxidase 5, STIM-dependent Ca2+ storage, and Fas-ligand induced apoptosis. Thus, obtaining a large amount of CaM is essential for our studies. In our laboratory, we expressed non-tagged CaM using a Lac operon in a pACYC vector and purified through a phenyl sepharose column, which typically gives low yields and limits the options for purification. We inserted a six-histidine tag to CaM and ligated it to a T7 promotor in pLW vector and purified tagged CaM through a Ni-NTA, which yields ~10 mg/L culture. We used a similar strategy for CaM-TnC chimeras, some of which could not previously be purified with phenyl sepharose. Furthermore, we prepared a CaM-immoblized affinity column to facilitate the purification of NOX5 protein.

Expression and purification of human Calmodulin derivates

243rd National Meeting (2012)

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