Original ArticlesExpression of Engineered Human I7β-Estradiol Dehydrogenase in a Prokaryotic System

OBJECTIVE:17β estradiol dehydrogenase (17βDH) is a model for pyridine-dependent steroid- converting enzymes. To define the structural and functional parameters of 17βDH, we created an expression system for production of abundant, homogeneous enzyme.METHODS:A full-length 17βDH cDNA clone was engineered into the inducible expression vector pMON 5839. After induction of plasmid-bearing Escherichia coli JM109 cells, the authenticity of the recombinant human placental 17βDH (r17βDH) was evaluated.RESULTS: Protein electrophoresis and Western blot analysis confirmed the immunologic identity of r17βDH with native human placental enzyme. The amino acid sequence, enzyme activity, Vmax, Km, and kcatof r17βDH matched that of the native enzyme.CONCLUSION:Prokaryotic cell lines offer the opportunity to create an unlimited supply of recombinant human placental 17βDH without the expense and time commitment of baculoviral or eukaryotic cell lines. We are now able to use r17βDH and its mutants to elucidate the mechanisms o...