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Journal of the Society for Gynecologic Investigation
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Original ArticlesExpression of Engineered Human I7ß-Estradiol Dehydrogenase in a Prokaryotic System

Donna L. O'Shea, MD

Departments of Obstetrics and Gynecology and Pediatrics, Washington University School of Medicine, St. Louis, Missouri

Michael J. Gast, MD, PhD

Departments of Obstetrics and Gynecology and Pediatrics, Washington University School of Medicine, St. Louis, Missouri

Gary L. Murdock, PhD

Departments of Obstetrics and Gynecology and Pediatrics, Washington University School of Medicine, St. Louis, Missouri

R. Mark Payne

Departments of Obstetrics and Gynecology and Pediatrics, Washington University School of Medicine, St. Louis, Missouri

Arnold W. Strauss, MD

Departments of Obstetrics and Gynecology and Pediatrics, Washington University School of Medicine, St. Louis, Missouri

OBJECTIVE: 17ß estradiol dehydrogenase (17ßDH) is a model for pyridine-dependent steroid- converting enzymes. To define the structural and functional parameters of 17ßDH, we created an expression system for production of abundant, homogeneous enzyme.

METHODS: A full-length 17ßDH 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 17ßDH (r17ßDH) was evaluated.

RESULTS: Protein electrophoresis and Western blot analysis confirmed the immunologic identity of r17ßDH with native human placental enzyme. The amino acid sequence, enzyme activity, Vmax, Km, and kcat of r17ßDH matched that of the native enzyme.

CONCLUSION: Prokaryotic cell lines offer the opportunity to create an unlimited supply of recombinant human placental 17ßDH without the expense and time commitment of baculoviral or eukaryotic cell lines. We are now able to use r17ßDH and its mutants to elucidate the mechanisms of action of this class of enzyme. (J Soc Gynecol Invest 1994;1:143-9)

Key Words: KEY WORDS: 17ß-estradiol dehydrogenase • protein expression • prokaryote expression systems • recombinant enzymes.

Journal of the Society for Gynecologic Investigation, Vol. 1, No. 2, 143-149 (1994)
DOI: 10.1177/107155769400100209
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