Abstract

Phosphodiesterase 3 enzymes (PDE3) play important roles in the regulation of adipocyte lipolysis and cardiac contractility by hydrolyzing cAMP and cGMP. This study reports the cloning, expression, and purification of recombinant Caenorhabditis elegans phosphodiesterase 3 (CEPDE3) isoforms, using a cold shock-based technique. The two closely related isoforms of the CEPDE3 gene (isoform F and isoform A) were cloned into the pGEX-6P-1 vector and expressed in E. coli as fusion proteins with a glutathione-S transferase (GST) tag at their amino terminus and purified by affinity chromatography using a glutathione Sepharose column. To optimize expression and recovery of soluble CEPDE3 protein from E. coli, we applied a “cold shock” technique at 4 °C following IPTG induction. Our findings suggest improved protein expression using an N-terminal GST tag instead of a C-terminal 6-histidine (6His) tag. Exposure of GST-tagged CEPDE3 to cold shock improved the protein solubility of CEPDE3 isoforms recovered by affinity chromatography. Studying CEPDE3 expression in C. elegans may enable us to understand the structure-function relationship and help crystallize the proteins to identify its catalytic pocket, contributing to the design of more effective small modulators of PDE3 catalytic activity.