Expression of recombinant phosphodiesterases 3A and 3B using baculovirus expression system

Abstract

Background: Phosphodiesterase 3A (PDE3A) and phosphodiesterase 3B (PDE3B) play a critical role in the regulation of intracellular level of adenosine 3´,5´-cyclic monophosphate (cyclic AMP, cAMP) and guanosine 3´,5´-cyclic monophosphate (cyclic GMP, cGMP). Subsequently PDE3 inhibitors have shown to relax vascular and inhibit platelet aggregation in cardiovascular disease. Objectives: In this study, our aim was to establish a method of expression for the recombinant human PDE3A and PDE3B proteins in insect cells using baculovirus expression system in order to investigate the activity of the expressed PDE3A and PDE3B proteins. Materials and Methods: The full length human PDE3A and PDE3B cDNA were cloned into recombinant baculovirus and transfected into the SF9 insect cells. Recombinant proteins were collected at 48 h, 60 h, 72 h, and 84 h post transfection. Transfection of recombinant baculovirus was verified by the morphological changes of the SF9 cells. Expression of human PDE3A and PDE3B was detected by using RT-PCR and western blot, respectively. The125I RIA method was used to determine the level of adenosine 3´,5´-cyclic monophosphate cAMP and cGMP, correspondingly. The activity of the expressed PDE3A and PDE3B proteins were investigated by cAMP and cGMP dsgradation with or without addition of milrinone, a potent and selective PDE inhibitor. Results: Recombinant human PDE3A and PDE3B proteins were stably expressed in SF9 cells and could be detected by distinct morphological changes in the SF9 cells, RT-PCR, and western blot at 48 h post-transfection. The molecular weights of the recombinant PDE3A and PDE3B molecular weights proteins were about 120 KDa and 135 KDa, respectively. Results of125I RIA assay showed that the levels of cAMP and cGMP were significantly decreased after incubation with the expressed PDE3A and PDE3B proteins. Furthermore, degradation of cAMP and cGMP through the activity of PDE3A and PDE3B was suppressed following to the addition of milrinone. Conclusions: Recombinant human PDE3A and PDE3B could be expressed in SF9 cells using baculovirus expression system, and thus provides the basic material for studying human PDE3A and PDE3B activity.