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• W2036829229 abstract "Soluble guanylate cyclase (sGC) has been purified from 100 L 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 100 mg 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 26 mg 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 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm 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 × 10−3 s−1 μM−1 that increased to 5.8 × 10−1 s−1 μM−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." @default.
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• W2036829229 date "2009-06-01" @default.
• W2036829229 modified "2023-10-14" @default.
• W2036829229 title "Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells" @default.
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• W2036829229 doi "https://doi.org/10.1016/j.pep.2009.01.001" @default.
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