Abstract
Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes that degrade the intracellular secondary messengers cyclic AMP (cAMP) and cyclic GMP (cGMP). The cGMP-binding cGMP-specific phosphodiesterase (PDE5) degrades cGMP and regulates the intracellular level of cGMP in many tissues including the smooth muscle of the corpus cavernosum. Sildenafi (Viagra) and Tadalafil (Cialis) are two specific PDE5 inhibitors, promote penile erection by blocking PDE5 activity, which causes cGMP accumulation in the corpus cavernosum and trigger smooth muscle relaxation and increases blood flow. In this study, a cDNA of thioredoxin- human PDE5 fusion protein was inserted into a cold-shock expression vector, pCold-PDE5A. This plasmid was transformed into E.coli strain BL21(DE3) and the recombinant protein was induced with 1mM IPTG at 15℃ for 24 hours. The soluble fraction of the fusion protein was purified by Ni2+-IDA affinity column. A simple method based on reverse-phase HPLC has been development for measuring the activity of recombinant human PDE5 protein. It allows quantization of product (GMP) and substrate (cGMP) in less than 15 min, and the sensitivity of detection is as low as 2x10-11 moles of GMP. The Km value of soluble human recombinant PDE5 protein is 1.12- 1.27 uM. Tadalafil (Cialis) potently inhibited the recombinant PDE5 protein with IC50 values of 39 nM, and the Ki value is 3.7 nM. The protein from inclusion bodies was also purified by on-column chemical refolding processes. However, the activity of recombinant Trx-PDE5 protein from inclusion bodies was not detected. We also established a method based on HPLC to detect two PDE5 inhibitors, Sildenafil and Tadalafil. Five different commercial drugs shown great inhibition of PDE5 activity were subjected to HPLC analysis, and it revealed that all of them contain either Sildenafil or Tadalafil.