Expression and Isolation of Recombinant sGC

Metabolic Radiolabeling of Recombinant Proteins in infected Insect Cells 

The insect cells were seeded in 2.5 ml of complete TClOO medium as described above. After the cells had attached, a viral inoculum (200-400 jj,l) was added to the cells. At the same time, a mixture of [ Sjmethionine and cysteine (0.5 mCi per 25-cm culture flask) was added to the medium. The time course of intracellular radioactive incorporation into total protein was analyzed over 72 hr. The cells were harvested and washed three times with cold PBS. The final cell pellet was resuspended in 300 /xl of lysis buffer, boiled for 5 min, and stored frozen at — 20°C for later analysis. Proteins were analyzed on 10% SDS-PAGE using 5-10 /jlI of the lysate. Radioactive incorporation into protein was detected after fluorography using EN HANCE (DuPont de Nemours) according to the manufacturer s protocol. Exposure of the dried gel on X-ray film was followed by laser densitomet-ric quantification. In parallel, after electrophoretic separation of the proteins, the gel was stained with Coomassie Brilliant Blue and dried, and the respective recombinant proteins were then cut out with a razor blade. Specific radioactive incorporation was measured by liquid scintillation counting. 

Supplementation of Recombinant sGC with Modified Heme Moieties 

5 mM phosphocreatine, and 5 U of creatine kinase. Enzymatic assays were started by addition of 4 mM MgCl2 and 1 mM GTP. After 10 min incubation at 37°C, the reaction was terminated by addition of 0.9 ml of 50 mM ice-cold sodium acetate buffer (pH 4.0) and heated at 90°C for 3 min. The amount of cGMP formed was determined by radioimmunoassay, as described previously (Kimura et al., 1975). 

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