Expression of engineered human 17 beta-estradiol dehydrogenase in a prokaryotic system.
OBJECTIVE
17 beta estradiol dehydrogenase (17 beta DH) is a model for pyridine-dependent steroid-converting enzymes. To define the structural and functional parameters of 17 beta DH, we created an expression system for production of abundant, homogeneous enzyme.


METHODS
A full-length 17 beta DH 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 17 beta DH (r17 beta DH) was evaluated.


RESULTS
Protein electrophoresis and Western blot analysis confirmed the immunologic identity of r17 beta DH with native human placental enzyme. The amino acid sequence, enzyme activity, Vmax, K(m), and kcat of r17 beta DH matched that of the native enzyme.


CONCLUSIONS
Prokaryotic cell lines offer the opportunity to create an unlimited supply of recombinant human placental 17 beta DH without the expense and time commitment of baculoviral or eukaryotic cell lines. We are now able to use r17 beta DH and its mutants to elucidate the mechanisms of action of this class of enzyme.
O'Shea
D L
DL
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri, USA.
Gast
M J
MJ
Murdock
G L
GL
Payne
R M
RM
Strauss
A W
AW
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000235:genetics; D004586:Electrophoresis; D004926:Escherichia coli; D004960:Estradiol Dehydrogenases / Q000096:biosynthesis / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis
D018076:DNA, Complementary; D011994:Recombinant Proteins; D004960:Estradiol Dehydrogenases