PRODUCTION OF DOG CALCYPHOSINE IN BACTERIA AND LACK OF PHOSPHORYLATION BY THE CATALYTIC SUBUNIT OF PROTEIN-KINASE-A IN-VITRO

Calcyphosine is a calcium-binding protein containing four EF-hand doma ins that is found in several epithelia and in some cells of the centra l nervous system. In thyroid follicular cells, calcyphosine is synthes ized and phosphorylated in response to stimulation by thyrotropin and cAMP agonists. The cDNA coding for dog calcyphosine has been expressed in bacteria under the control of the T7 promoter. Recombinant calcyph osine was purified from crude bacterial lysates by a combination of an ion-exchange and hydrophobic interaction chromatography. Phosphorylati on assays using the purified catalytic subunit of protein kinase A and the recombinant or the native calcyphosine revealed that, contrary to a previous report, calcyphosine is not significantly phosphorylated b y this enzyme in vitro. (C) 1997 Elsevier Science Ireland Ltd.