Secretory expression, purification and functional characterization of 17β-hydroxysteroid dehydrogenase type 1 from mammalian HEK293T cells.

Chen, J ; Feng, W ; et al.

In: Protein expression and purification, Jg. 137 (2017-09-01), S. 52-57

academicJournal

17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) mainly catalyzes the reduction of estrone into estradiol. The enzymatic conversion is a critical step in estradiol accumulation in breast tissue, which is a valuable prognosis index of breast cancer disease. However, the source of 17β-HSD1 for inhibitor design is limited. In this study, the fragment encoding human 17β-HSD1 was successfully cloned and expressed in human embryonic kidney (HEK) 293T mammalian cells. The recombinant protein was purified by immobilized metal ion affinity chromatography yielding above 17 mg of purified 17β-HSD1 protein per liter of cell culture, with a specific activity of 8.54 μmoL/min/mg of protein for conversion of estradiol into estrone, with NAD + as cofactor at pH 9.2. Enzyme characterization studies revealed that the protein has estrogenic activity and the K m value for estrone is about 20 nM. The recombinant protein purified from transfected HEK293T cells had higher specific activity compared to that of the enzyme purified directly from placenta. The present data show that the mammalian cell expression system can provide active 17β-HSD1 which is functionally identical to its natural counterpart and easy to purify in qualities suitable for its structure-function study.

Titel:	Secretory expression, purification and functional characterization of 17β-hydroxysteroid dehydrogenase type 1 from mammalian HEK293T cells.
Autor/in / Beteiligte Person:	Chen, J ; Feng, W ; Zhao, Y
Zeitschrift:	Protein expression and purification, Jg. 137 (2017-09-01), S. 52-57
Veröffentlichung:	Orlando, FL : Academic Press ; <i>Original Publication</i>: San Diego : Academic Press, c1990-, 2017
Medientyp:	academicJournal
ISSN:	1096-0279 (electronic)
DOI:	10.1016/j.pep.2017.06.015
Schlagwort:	Chromatography, Affinity methods HEK293 Cells Humans Recombinant Proteins biosynthesis Recombinant Proteins chemistry Recombinant Proteins genetics Recombinant Proteins isolation & purification Substrate Specificity 17-Hydroxysteroid Dehydrogenases biosynthesis 17-Hydroxysteroid Dehydrogenases chemistry 17-Hydroxysteroid Dehydrogenases genetics 17-Hydroxysteroid Dehydrogenases isolation & purification Estrone chemistry Gene Expression
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article Language: English [Protein Expr Purif] 2017 Sep; Vol. 137, pp. 52-57. <i>Date of Electronic Publication: </i>2017 Jun 29. MeSH Terms: 17-Hydroxysteroid Dehydrogenases* / biosynthesis ; 17-Hydroxysteroid Dehydrogenases* / chemistry ; 17-Hydroxysteroid Dehydrogenases* / genetics ; 17-Hydroxysteroid Dehydrogenases* / isolation & purification ; Gene Expression* ; Estrone / *chemistry ; Chromatography, Affinity / methods ; HEK293 Cells ; Humans ; Recombinant Proteins / biosynthesis ; Recombinant Proteins / chemistry ; Recombinant Proteins / genetics ; Recombinant Proteins / isolation & purification ; Substrate Specificity Contributed Indexing: Keywords: 17β-HydroxySteroid dehydrogenase; Enzyme kinetic; Estradiol; Estrone; Transient expression Substance Nomenclature: 0 (Recombinant Proteins) ; 2DI9HA706A (Estrone) ; EC 1.1.- (17-Hydroxysteroid Dehydrogenases) ; EC 1.1.1.51 (3 (or 17)-beta-hydroxysteroid dehydrogenase) Entry Date(s): Date Created: 20170704 Date Completed: 20170807 Latest Revision: 20170807 Update Code: 20231215

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