The engineered, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase: purification, characterization and crystallization
2001
Human type I 3-hydroxysteroid dehydrogenase/ isomerase (3-HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membranespanning domain (residues 283‐310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3-HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD + and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38·8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42·0 kDa). Michaelis‐Menten constants measured for 3-HSD substrate (dehydroepiandrosterone) utilization by
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