Refolding and kinetic characterization of the phosphodiesterase-8A catalytic domain.

Cyclic nucleotide phosphodiesterase-8 (PDE8) hydrolyzes the second messenger cAMP and is involved in many biological processes such as testosterone production. Although the bacterial and mammalian expression systems have been extensively tried, production of large quantity of soluble and active PDE8 remains to be a major hurdle for pharmacological and structural studies. Reported here is a detailed protocol of refolding and purification of large quantity of the PDE8A1 catalytic domain (residues 480–820) and kinetic characterization of the refolded protein. This protocol yielded about 8mg of the PDE8A catalytic domain from 2l Escherichia coli culture, which has at least 40-fold higher activity than those reported in literature. The PDE8A1 catalytic domain has kcat of 4.0s−1 for Mn2+ and 2.9s−1 for Mg2+, and the KM values of 1–1.8μM. In addition, the PDE8A1 (205–820) fragment that contains both PAS and catalytic domains was expressed in E. coli and refolded. This PDE8A1 (205–820) fragment has kcat of 1.1s−1 and KM of 0.28μM, but aggregated at high concentration. The KM of PDE8A1 (205–820) is 2- to 7-fold higher than the KM values of 40–150nM for the full-length PDE8s in literature, but about 6-fold lower than that of the catalytic domain. Thus, the KM difference likely implies an allosteric regulation of the PDE8A activity by its PAS domain.