Analysis of single-stranded cAMP-response element binding protein by glutathione-S-transferase fusion protein expression system

We reported previously that the binding activity of a nuclear protein (ssCRE-BP) to single-stranded CRE is altered by long-term treatment with morphine in the mouse cerebellum. The cloning and sequencing of a cDNA encoding ssCRE-BP showed that the protein possesses a glycine-rich domain and a glutamine-rich domain in the amino terminus and the carboxyl terminus, respectively. To investigate the region of ssCRE-BP involved in DNA binding activity, recombinant glutathione-S-transferase (GST) fusion proteins containing ssCRE-BP were expressed in bacterial systems. Deletion of carboxyl terminal region of ssCRE-BP decreased the DNA binding activity of the protein, suggesting that glutamine-rich domain is required for the binding of ssCRE-BP to single-stranded CRE. Rabbit anti-ssCRE-BP antibodies were raised against a GST-ssCRE-BP fusion protein. Using the antibodies in western blot analysis, a polypeptide of ～38 kDa was detected in the brain. These findings indicate that the GST-ssCRE-BP fusion proteins can be a useful tool to investigate the function of ssCRE-BP in opiate tolerance and dependence.