Original ArticlesExpression of Engineered Human I7β-Estradiol Dehydrogenase in a Prokaryotic System
| Autor: | G L Murdock, R M Payne, D L O'Shea, A W Strauss, M J Gast |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
chemistry.chemical_classification
Expression vector medicine.diagnostic_test Obstetrics and Gynecology Biology medicine.disease_cause Molecular biology Enzyme assay law.invention Enzyme Biochemistry chemistry Western blot law Recombinant DNA medicine biology.protein Beta (finance) Escherichia coli Peptide sequence |
| Zdroj: | Journal of the Society for Gynecologic Investigation. 1:143-149 |
| ISSN: | 1071-5576 |
| DOI: | 10.1177/107155769400100209 |
| Popis: | 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. Conclusion 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. |
| Databáze: | OpenAIRE |
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