Human soluble guanylate cyclase: Functional expression, purification and structural characterization

Soluble guanylate cyclase is an enzyme that catalyzes formation of cGMP fro m GTP and is a member of the nucleotide cyclase family of enzymes. sGC is a receptor for endogenous and exogenous nitric oxide and is activated severa l-fold upon its binding, constituting a core enzyme in the nitric oxide sig nal transduction pathway. cGMP generated by sGC is an important second mess enger that regulates activity of several enzymes triggering such important physiologic reactions as vasodilation, smooth muscle relaxation and platele t aggregation. We report here the functional expression of the human isofor m of soluble guanylate cyclase in HighFive insect cells using a baculovirus expression system. Highly active recombinant protein was obtained without heme reconstitution or supplementation of the cell growth medium and the le vel of protein expression was found to be heavily affected by the compositi on of the growth medium. We have successfully purified highly active sGC (s p act up to 940 nmol/min/mg) from adherent cultures using a three-column, 1 -day procedure. The UV-Vis spectrum of the isolated protein shows a Soret b and at 431 nm, consistent with a histidine-ligated, 5-coordinate heme as pr eviously reported. Far UV CD spectroscopy, intrinsic tryptophan fluorescenc e, fluorescence of the hydrophobic dye bis-ANS, size-exclusion chromatograp hy, and small angle X-ray scattering (SAXS) were used to characterize the s tructural properties of the purified sGC. We used two hierarchical neural n etwork methods to predict the secondary structure of sGC and found it to be consistent with the observed CD spectrum of sGC. (C) 2001 Academic Press.