BAFF Receptor mAb Treatment Ameliorates Development and Progression of Atherosclerosis in Hyperlipidemic ApoE?/? Mice

Aims

Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE^−/− mouse.

Methods and Results

To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE^−/− mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93⁻ CD19⁺ B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19⁺ B cells, CD4⁺ and CD8⁺ T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE^−/− mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93⁻ CD19⁺ B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment.

Conclusion

Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE^−/− mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.     

Tissue-Specific Increases in 11β-Hydroxysteroid Dehydrogenase Type 1 in Normal Weight Postmenopausal Women

With age and menopause there is a shift in adipose distribution from gluteo-femoral to abdominal depots in women. Associated with this redistribution of fat are increased risks of type 2 diabetes and cardiovascular disease. Glucocorticoids influence body composition, and 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) which converts inert cortisone to active cortisol is a putative key mediator of metabolic complications in obesity. Increased 11βHSD1 in adipose tissue may contribute to postmenopausal central obesity. We hypothesized that tissue-specific 11βHSD1 gene expression and activity are up-regulated in the older, postmenopausal women compared to young, premenopausal women. Twenty-three pre- and 23 postmenopausal, healthy, normal weight women were recruited. The participants underwent a urine collection, a subcutaneous adipose tissue biopsy and the hepatic 11βHSD1 activity was estimated by the serum cortisol response after an oral dose of cortisone. Urinary (5α-tetrahydrocortisol+5β-tetrahydrocortisol)/tetrahydrocortisone ratios were higher in postmenopausal women versus premenopausal women in luteal phase (P<0.05), indicating an increased whole-body 11βHSD1 activity. Postmenopausal women had higher 11βHSD1 gene expression in subcutaneous fat (P<0.05). Hepatic first pass conversion of oral cortisone to cortisol was also increased in postmenopausal women versus premenopausal women in follicular phase of the menstrual cycle (P<0.01, at 30 min post cortisone ingestion), suggesting higher hepatic 11βHSD1 activity. In conclusion, our results indicate that postmenopausal normal weight women have increased 11βHSD1 activity in adipose tissue and liver. This may contribute to metabolic dysfunctions with menopause and ageing in women.     

Substituted phenyl triazoles as selective inhibitors of 11β-Hydroxysteroid Dehydrogenase Type 1

3-(Phenylcyclobutyl)-1,2,4-triazoles were identified as inhibitors of 11β-Hydroxysteroid Dehydrogenase Type 1 (HSD1). They were shown to be active in the mouse in vivo pharmacodynamic model (PD) for HSD1 but exhibited a potent off-target activation of the Pregnane X Receptor (PXR). SAR studies and synthesis of analogs that led to the discovery of a selective HSD1 inhibitor are described in detail.     

Molecular Framework of Steroid/Retinoid Discrimination in 17β-Hydroxysteroid Dehydrogenase Type 1 and Photoreceptor-associated Retinol Dehydrogenase

Steroids and retinoids are signaling molecules that control a variety of physiological processes. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of estrone to estradiol, supplying biologically active estrogen-regulating sex-specific differentiation. Photoreceptor-associated retinol dehydrogenase (prRDH) is evolutionarily closely related to 17β-HSD1 but reduces all-trans retinal to all-trans retinol, contributing to rhodopsin regeneration in the visual cycle. Sequence alignment revealed a new enzyme-specific conserved amino acid close to the active site: methionine (position 144 in human enzyme) in prRDH and glycine (position 145) in 17β-HSD1. We investigated the role of this residue in substrate discrimination in human and zebrafish enzymes. Both recombinant enzymes were expressed in HEK 293 cells followed by normalization of expression by semiquantitative Western blots. Changing of the prRDH-specific methionine to glycine resulted in a gain of function: the mutants now catalyzed the reduction of estrone and all-trans retinal. Human and zebrafish wild-type 17β-HSD1s efficiently catalyzed the reduction of all-trans retinal to its alcohol. Exchange of glycine for methionine increased the catalytic activity of 17β-HSD1 toward all-trans retinal in zebrafish but not in the human enzyme, in which the opposite effect was observed. Molecular modeling showed that the zebrafish 17β-HSD1 substrate-binding pocket is similar to that of prRDH and methionine insertion benefits all-trans retinal reduction. In contrast, in human 17β-HSD1, the insertion of the bulky methionine causes a disruption of substrate-binding site. We demonstrate for the first time the role of a single amino acid in the evolution of these functionally diverse enzymes and suggest new physiological functions for 17β-HSD1 in retinoid metabolism. This has implications for the validation of inhibitors of 17β-HSD1 developed for cancer treatment.     

Differential Regulation of 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Patients with Differing Etiologies of Hypopituitarism

Objective: Pituitary patients with different etiologies of hypopituitarism exhibit differing phenotypes, despite similar replacement therapy strategies. We hypothesized that differential regulation of the isoenzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which mediates the net autocrine conversion of cortisone to cortisol in adipose tissues and liver, may play a role.Methods: We studied 11β-HSD1 activity (using urine cortisol/cortisone metabolites ratio) in 36 hypopituitary patients with treated craniopharyngiomas, treated remitted Cushing disease, and treated nonfunctioning pituitary adenomas + prolactinomas on and off growth hormone (GH) replacement.Results: 11β-HSD1 activity was higher in subjects with craniopharyngioma both on and off GH, as evidenced by increased tetrahydrocortisol to tetrahydrocortisone metabolite ratios compared to other diagnostic groups, but there was no difference in body mass index, insulin levels, serum hormone measurements, or hydrocortisone dose between groups.Conclusion: Craniopharyngiomas are associated with enhanced 11β-HSD1 activity compared to other diagnostic hypopituitary groups, and this may contribute to the adverse phenotypic and metabolic features seen in this condition.Abbreviations: BMI = body mass index; Em = cortisone metabolites; Fm = cortisol metabolites; GH = growth hormone; 11β-HSD1 = 11β-hydroxysteroid dehydrogenase type 1; IGF-1 = insulin-like growth factor 1; NFPA = nonfunctioning pituitary adenoma; THE = tetrahydrocortisone; THF = tetrahydrocortisol     

Interactions of 17β-Hydroxysteroid Dehydrogenase Type 10 and Cyclophilin D in Alzheimer's Disease

The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (k_a 2.0?×?10⁵ M¹s^?1, k_d 5.8?×?10⁴ s^?1, and K_D 3.5?×?10^–10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10–cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10–cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10–cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.

     

Dihydrocapsaicin Attenuates Plaque Formation through a PPARγ/LXRα Pathway in apoE?/? Mice Fed a High-Fat/High-Cholesterol Diet

Aims

Atherosclerosis is a chronic inflammatory disease and represents the major cause of cardiovascular morbidity and mortality. There is evidence that dihydrocapsaicin (DHC) can exert multiple pharmacological and physiological effects. Here, we explored the effect of DHC in atherosclerotic plaque progression in apoE^−/− mice fed a high-fat/high-cholesterol diet.

Methods and Results

apoE^−/− mice were randomly divided into two groups and fed a high-fat/high-cholesterol diet with or without DHC for 12 weeks. We demonstrated that cellular cholesterol content was significantly decreased while apoA1-mediated cholesterol efflux was significantly increased following treatment with DHC in THP-1 macrophage-derived foam cells. We also observed that plasma levels of TG, LDL-C, VLDL-C, IL-1β, IL-6, TNF-α and CRP were markedly decreased while plasma levels of apoA1 and HDL-C were significantly increased, and consistent with this, atherosclerotic lesion development was significantly inhibited by DHC treatment of apoE^−/− mice fed a high-fat/high-cholesterol diet. Moreover, treatment with both LXRα siRNA and PPARγ siRNA made the up-regulation of DHC on ABCA1, ABCG1, ABCG5, SR-B1, NPC1, CD36, LDLR, HMGCR, apoA1 and apoE expression notably abolished while made the down-regulation of DHC on SRA1 expression markedly compensated. And treatment with PPARγ siRNA made the DHC-induced up-regulation of LXRα expression notably abolished while treatment with LXRα siRNA had no effect on DHC-induced PPARγ expression.

Conclusion

These observations provide direct evidence that DHC can significantly decrease atherosclerotic plaque formation involving in a PPARγ/LXRα pathway and thus DHC may represent a promising candidate for a therapeutic agent for the treatment or prevention of atherosclerosis.     

Development of a High-Throughput Cell-Based Assay for 11β-Hydroxysteroid Dehydrogenase Type 1 Using BacMam Technology

Cortisol is an important glucocorticoid in humans that regulates many physiological processes. Human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone to cortisol in vivo and has emerged as an appealing therapeutic target for treating metabolic diseases. Here, we report a sensitive and robust high-throughput (HT) cell-based assay for screening 11beta-HSD1 inhibitors. This assay utilizes a HEK293 cell line transduced by a BacMam virus expressing human 11beta-HSD1. The enzyme activity in the cells was measured by quantifying cortisol levels released into the cell culture supernatant via a competitive homogenous time-resolved fluorescence (HTRF) method. We show that 11beta-HSD1 activity in supernatant of BacMam-transduced HEK293 cells increases with 11beta-HSD1 BacMam virus load in a dose-dependent manner, and is comparable to the enzyme activity detected in differentiated mouse adipocytes. In addition, we show that co-expression of hexose-6-phosphate dehydrogenase (H6PDH) is not required for the enzyme to function effectively as an oxo-reductase. This assay has been developed in low-volume 384-well format and it is sensitive, robust, and amenable to HT screening.     

α Cell Function and Gene Expression Are Compromised in Type 1 Diabetes

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Curcumin as a Potent and Selective Inhibitor of 11β-Hydroxysteroid Dehydrogenase 1: Improving Lipid Profiles in High-Fat-Diet-Treated Rats

Background

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activates glucocorticoid locally in liver and fat tissues to aggravate metabolic syndrome. 11β-HSD1 selective inhibitor can be used to treat metabolic syndrome. Curcumin and its derivatives as selective inhibitors of 11β-HSD1 have not been reported.

Methodology

Curcumin and its 12 derivatives were tested for their potencies of inhibitory effects on human and rat 11β-HSD1 with selectivity against 11β-HSD2. 200 mg/kg curcumin was gavaged to adult male Sprague-Dawley rats with high-fat-diet-induced metabolic syndrome for 2 months.

Results and Conclusions

Curcumin exhibited inhibitory potency against human and rat 11β-HSD1 in intact cells with IC₅₀ values of 2.29 and 5.79 µM, respectively, with selectivity against 11β-HSD2 (IC₅₀, 14.56 and 11.92 µM). Curcumin was a competitive inhibitor of human and rat 11β-HSD1. Curcumin reduced serum glucose, cholesterol, triglyceride, low density lipoprotein levels in high-fat-diet-induced obese rats. Four curcumin derivatives had much higher potencies for Inhibition of 11β-HSD1. One of them is (1E,4E)-1,5-bis(thiophen-2-yl) penta-1,4-dien-3-one (compound 6), which had IC₅₀ values of 93 and 184 nM for human and rat 11β-HSD1, respectively. Compound 6 did not inhibit human and rat kidney 11β-HSD2 at 100 µM. In conclusion, curcumin is effective for the treatment of metabolic syndrome and four novel curcumin derivatives had high potencies for inhibition of human 11β-HSD1 with selectivity against 11β-HSD2.     

Decreased 11β-Hydroxysteroid Dehydrogenase 1 Level and Activity in Murine Pancreatic Islets Caused by Insulin-Like Growth Factor I Overexpression

We have reported a high expression of IGF-I in pancreatic islet β-cells of transgenic mice under the metallothionein promoter. cDNA microarray analysis of the islets revealed that the expression of 82 genes was significantly altered compared to wild-type mice. Of these, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which is responsible for the conversion of inert cortisone (11-dehydrocorticosterone, DHC in rodents) to active cortisol (corticosterone) in the liver and adipose tissues, has not been identified previously as an IGF-I target in pancreatic islets. We characterized the changes in its protein level, enzyme activity and glucose-stimulated insulin secretion. In freshly isolated islets, the level of 11β-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence, 11β-HSD1 was observed exclusively in glucagon-producing, islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced enzymatic activities. Dexamethasone (DEX) and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice, 48-h pre-incubation of DEX caused a significant decrease in insulin release, while the effect of DHC was largely blunted consistent with diminished 11β-HSD1 activity. In order to establish the function of intracrine glucocorticoids, we overexpressed 11β-HSD1 cDNA in MIN6 insulinoma cells, which together with DHC caused apoptosis and a significant decrease in proliferation. Both effects were abolished with the treatment of an 11β-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11β-HSD1 expression and activity within the pancreatic islets, which may mediate part of the IGF-I effects on cell proliferation, survival and insulin secretion.     

Oral Supplementation with Non-Absorbable Antibiotics or Curcumin Attenuates Western Diet-Induced Atherosclerosis and Glucose Intolerance in LDLR?/? Mice – Role of Intestinal Permeability and Macrophage Activation

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as Type 2 Diabetes and atherosclerosis) has shifted the focus from Western diet-induced changes in gut microbiota per se to release of gut bacteria-derived products into circulation as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. Under physiological conditions, an intact intestinal barrier prevents this release of LPS underscoring the importance of examining and modulating the direct effects of Western diet on intestinal barrier function. In the present study we evaluated two strategies, namely selective gut decontamination and supplementation with oral curcumin, to modulate Western-diet (WD) induced changes in intestinal barrier function and subsequent development of glucose intolerance and atherosclerosis. LDLR−/− mice were fed WD for 16 weeks and either received non-absorbable antibiotics (Neomycin and polymyxin) in drinking water for selective gut decontamination or gavaged daily with curcumin. WD significantly increased intestinal permeability as assessed by in vivo translocation of FITC-dextran and plasma LPS levels. Selective gut decontamination and supplementation with curcumin significantly attenuated the WD-induced increase in plasma LPS levels (3.32 vs 1.90 or 1.51 EU/ml, respectively) and improved intestinal barrier function at multiple levels (restoring intestinal alkaline phosphatase activity and expression of tight junction proteins, ZO-1 and Claudin-1). Consequently, both these interventions significantly reduced WD-induced glucose intolerance and atherosclerosis in LDLR−/− mice. Activation of macrophages by low levels of LPS (50 ng/ml) and its exacerbation by fatty acids is likely the mechanism by which release of trace amounts of LPS into circulation due to disruption of intestinal barrier function induces the development of these diseases. These studies not only establish the important role of intestinal barrier function, but also identify oral supplementation with curcumin as a potential therapeutic strategy to improve intestinal barrier function and prevent the development of metabolic diseases.     

Celiac Disease–Specific TG2-Targeted Autoantibodies Inhibit Angiogenesis Ex Vivo and In Vivo in Mice by Interfering with Endothelial Cell Dynamics

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility.     

Folic Acid Supplementation during Pregnancy Induces Sex-Specific Changes in Methylation and Expression of Placental 11β-Hydroxysteroid Dehydrogenase 2 in Rats

In the placenta, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA) during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg) or normal low (1mg/kg, control) levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (−378 to −275) was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11β-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%). This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.     

Protective Effects of PARP-1 Knockout on Dyslipidemia-Induced Autonomic and Vascular Dysfunction in ApoE?/? Mice: Effects on eNOS and Oxidative Stress

The aims of this study were to investigate the role of poly(ADP-ribose) polymerase (PARP)-1 in dyslipidemia-associated vascular dysfunction as well as autonomic nervous system dysregulation. Apolipoprotein (ApoE)^−/− mice fed a high-fat diet were used as a model of atherosclerosis. Vascular and autonomic functions were measured in conscious mice using telemetry. The study revealed that PARP-1 plays an important role in dyslipidemia-associated vascular and autonomic dysfunction. Inhibition of this enzyme by gene knockout partially restored baroreflex sensitivity in ApoE^−/− mice without affecting baseline heart-rate and arterial pressure, and also improved heart-rate responses following selective blockade of the autonomic nervous system. The protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction was associated with preservation of eNOS activity. Dyslipidemia induced PARP-1 activation was accompanied by oxidative tissue damage, as evidenced by increased expression of iNOS and subsequent protein nitration. PARP-1 gene deletion reversed these effects, suggesting that PARP-1 may contribute to vascular and autonomic pathologies by promoting oxidative tissue injury. Further, inhibition of this oxidative damage may account for protective effects of PARP-1 gene deletion on vascular and autonomic functions. This study demonstrates that PARP-1 participates in dyslipidemia-mediated dysregulation of the autonomic nervous system and that PARP-1 gene deletion normalizes autonomic and vascular dysfunctions. Maintenance of eNOS activity may be associated with the protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction.     

Rescue of a Recombinant Machupo Virus from Cloned cDNAs and In Vivo Characterization in Interferon (αβ/γ) Receptor Double Knockout Mice

Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.     

Expression,Purification, and Characterization of a Human Recombinant 17β-Hydroxysteroid Dehydrogenase Type 1 in <Emphasis Type=Italic>Escherichia coli</Emphasis>

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reaction of estrone with NADPH to form estradiol and NADP⁺, thereby regulating the biological activity of sex steroid hormones in a variety of tissues. Here, we present an efficient method for expressing and purifying human 17β-HSD1 from Escherichia coli. The expression vector pET28a/17β-HSD1 was constructed and transformed into Escherichia coli BL21(DE3) cells. We found that the active enzyme can be obtained by inducing 17β-HSD1 expression at 0.25 mM IPTG, 13°C for overnight. The protein is purified by single step Ni–NTA affinity chromatography and yields 2.8 mg/L of culture. The kinetic study shows V/E _t of (1.21 ± 0.05) × 10⁻²/s and K _estradiol of 0.8 μM in the oxidation of estradiol with NADP⁺ as cofactor at pH 9.3. The new bacterial expression system for recombinant 17β-HSD1 is useful for the easy purification of large amounts and will facilitate the functional study of this enzyme.