Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells

Soluble guanylate cyclase (sGC) has been purified from 100L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni2+-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a Mr=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS–PAGE corresponding to the α (Mr ∼77,000) and β (Mr ∼70,000) sGC subunits. It showed an A430/A280=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had kcat/Km=1.7×10−3s−1μM−1 that increased to 5.8×10−1s−1μM−1 upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure–function relationships.