Expression and binding studies of phosphodiesterase 4A5 with binding partner AIP

Abstract

The PDE4A5/AIP complex is a regulatory complex in the pituitary gland and loss of function of this complex, due to an AIP mutation, has been identified in 20% of patients with familial isolated pituitary adenoma. This study aims to test different PDE4A5 expression protocols, attempt to improve PDE4A5 solubility, test different purification protocols for PDE4A5 and attempt to generate the PDE4A5/AIP complex. Following generation of master plates containing transformed BL21 cells with the pETite H6-SUMO-PDE vector, optimisation of expression and induction protocols were carried out and a successful expression protocol was obtained. These cells were lysed with numerous conditions, but all failed to show a large marked improvement in the solubility of PDE4A5 even after the addition of additives to one of the best performing buffers. Soluble PDE4A5 purification using HIS-tag chromatography was attempted, which yielded PDE4A5 with a 69% purity. On column re-folding HIS-tag chromatography for PDE4A5 present as an inclusion body, yielded a solubilised PDE4A5 with a 76% purity which however lacked secondary structure as evidenced by circular dichroism analyses. Contrastingly, PDE4A5 purified from the soluble fraction, showed an ordered secondary structure and following CDSSTR analyses, a predominately helical protein structure was predicted. The soluble PDE4A5 was used for binding studies using blue native gels which did not yield the PDE4A5/AIP complex. A homology model for 59% of the total PDE4A5 was also generated. Thus, in conclusion, further optimisation of PDE4A5 solubility and purification may be attempted alongside new binding conditions in order to generate the PDE4A5/AIP complex.