Human soluble guanylate cyclase: functional expression, purification and structural characterization.
Soluble guanylate cyclase is an enzyme that catalyzes formation of cGMP from 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 several-fold upon its binding, constituting a core enzyme in the nitric oxide signal transduction pathway. cGMP generated by sGC is an important second messenger that regulates activity of several enzymes triggering such important physiologic reactions as vasodilation, smooth muscle relaxation and platelet aggregation. We report here the functional expression of the human isoform 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 level of protein expression was found to be heavily affected by the composition of the growth medium. We have successfully purified highly active sGC (sp 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 band at 431 nm, consistent with a histidine-ligated, 5-coordinate heme as previously reported. Far UV CD spectroscopy, intrinsic tryptophan fluorescence, fluorescence of the hydrophobic dye bis-ANS, size-exclusion chromatography, and small angle X-ray scattering (SAXS) were used to characterize the structural properties of the purified sGC. We used two hierarchical neural network methods to predict the secondary structure of sGC and found it to be consistent with the observed CD spectrum of sGC.
Kosarikov
D N
DN
Department of Chemistry and Biochemistry, San Francisco State University, California, USA.
Young
P
P
Uversky
V N
VN
Gerber
N C
NC
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013057:Spectrum Analysis; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D006162:Guanylate Cyclase