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• W1988205364 abstract "Human membrane 17β-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17β-hydroxysteroids into their inactive keto forms in a variety of tissues. 17β-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-β-d-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel–chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-β-d-maltoside-destabilized liposomes prepared from l-α-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 μmol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 μmol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 ± 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17β-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K m of NAD being 110 ± 10 μm and that of NADP 9600 ± 100 μm using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20α-dihydroprogesterone as substrates were also determined. Human membrane 17β-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17β-hydroxysteroids into their inactive keto forms in a variety of tissues. 17β-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-β-d-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel–chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-β-d-maltoside-destabilized liposomes prepared from l-α-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 μmol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 μmol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 ± 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17β-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K m of NAD being 110 ± 10 μm and that of NADP 9600 ± 100 μm using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20α-dihydroprogesterone as substrates were also determined. 17β-hydroxysteroid dehydrogenase dodecyl-β-d-maltoside bis-sulfosuccinimidyl suberate l-α-phosphatidylcholine dithiothreitol androstenedione The members of the 17β-hydroxysteroid dehydrogenase (17β-HSD)1 family are crucial in the biosynthesis and metabolism of active steroid hormones in a variety of tissues. Estrogens and androgens in turn control a variety of important physiological functions such as growth, reproduction, and differentiation. Using NAD as cofactor, 17β-HSD2, with its predominantly oxidative activity, primarily converts the highly active 17β-hydroxysteroids such as estradiol, testosterone, and dihydrotestosterone into their inactive keto forms. Furthermore, studies carried out in vitro indicate that 17β-HSD2 is able to use C20-steroids as substrates, namely to catalyze the oxidation of 20α-dihydroprogesterone to progesterone. The expression of the mRNA of human 17β-HSD2 has been detected in a large variety of tissues. Its 1.5-kb mRNA is highly expressed in the endometrium, placenta, liver, and small intestine and also in smaller amounts in the pancreas, colon, kidney, and prostate (1Wu L. Einstein M. Geissler W.M. Chan H.K. Elliston K.O. Andersson S. J. Biol. Chem. 1993; 268: 12964-12969Abstract Full Text PDF PubMed Google Scholar, 2Cassey M.L. MacDonald P.C. Andersson S. J. Clin. Invest. 1994; 94: 2135-2141Crossref PubMed Scopus (193) Google Scholar, 3Miettinen M.M. Mustonen M.V.J. Poutanen M.H. Isomaa V.V. Vihko R.K. Biochem. J. 1996; 314: 839-845Crossref PubMed Scopus (176) Google Scholar, 4Mustonen M. Poutanen M. Chotteau-Lelievre A. deLaunoit Y. Isomaa V. Vainio S. Vihko R. Vihko P. Mol. Cell. Endocrinol. 1997; 134: 33-40Crossref PubMed Scopus (26) Google Scholar, 5Mustonen M.V.J. Poutanen M.H. Kellokumpu S. deLaunoit Y. Isomaa V.V. Vihko R.K. Vihko P.T. J. Mol. Endocrinol. 1998; 20: 67-74Crossref PubMed Scopus (47) Google Scholar). Human 17β-HSD2 mRNA has also been found to be present in human breast, endometrial, and prostate cancer cell lines (3Miettinen M.M. Mustonen M.V.J. Poutanen M.H. Isomaa V.V. Vihko R.K. Biochem. J. 1996; 314: 839-845Crossref PubMed Scopus (176) Google Scholar). In addition, both rodent and human 17β-HSD2 enzymes are widely distributed in the gastrointestinal and urinary tracts, in the liver, as well as in the adrenals of adults and developing fetuses (2Cassey M.L. MacDonald P.C. Andersson S. J. Clin. Invest. 1994; 94: 2135-2141Crossref PubMed Scopus (193) Google Scholar, 3Miettinen M.M. Mustonen M.V.J. Poutanen M.H. Isomaa V.V. Vihko R.K. Biochem. J. 1996; 314: 839-845Crossref PubMed Scopus (176) Google Scholar, 4Mustonen M. Poutanen M. Chotteau-Lelievre A. deLaunoit Y. Isomaa V. Vainio S. Vihko R. Vihko P. Mol. Cell. Endocrinol. 1997; 134: 33-40Crossref PubMed Scopus (26) Google Scholar, 5Mustonen M.V.J. Poutanen M.H. Kellokumpu S. deLaunoit Y. Isomaa V.V. Vihko R.K. Vihko P.T. J. Mol. Endocrinol. 1998; 20: 67-74Crossref PubMed Scopus (47) Google Scholar). Recently, the correlation between 17β-HSD2 and colonic cancer was reported (6English M.A. Stewart P.M. Hewison M. Mol. Cell. Endocrinol. 2001; 171: 53-60Crossref PubMed Scopus (32) Google Scholar). The broad tissue distribution, together with the predominant oxidative activity of 17β-HSD2, suggests that the enzyme plays an essential role in the inactivation of highly active 17β-hydroxysteroids. It may have a protective role by lowering the active steroid concentrations and reducing excessive sex hormone action in target tissues. 17β-HSD2 is a trans-membrane protein, which is demonstrated by its subcellular distribution in the endoplasmic reticulum (7Puranen T.J. Kurkela R.M. Lakkakorpi J.T. Poutanen M.H. Itaranta P.V. Melis J.P. Ghosh D. Vihko R.K. Vihko P.T. Endocrinology. 1999; 140: 3334-3341Crossref PubMed Scopus (49) Google Scholar). 17β-HSD2 cDNA encodes a predicted protein of 387 amino acids with a molecular mass of 42,782 daltons. The primary structure shows that it belongs to the type II signal anchor membrane protein, which is characterized by possessing a cluster of positively charged amino acids and followed by a hydrophobic core of about 33 nonpolar amino acids close to the N terminus of the protein (1Wu L. Einstein M. Geissler W.M. Chan H.K. Elliston K.O. Andersson S. J. Biol. Chem. 1993; 268: 12964-12969Abstract Full Text PDF PubMed Google Scholar, 8von Heijne G. Gavel Y. Eur. J. Biochem. 1988; 174: 671-678Crossref PubMed Scopus (572) Google Scholar, 9von Heijne G. Nature. 1989; 341: 456-458Crossref PubMed Scopus (434) Google Scholar). The carboxyl terminus has a luminal carboxyl-terminal endoplasmic reticulum retention motif (KKK) (1Wu L. Einstein M. Geissler W.M. Chan H.K. Elliston K.O. Andersson S. J. Biol. Chem. 1993; 268: 12964-12969Abstract Full Text PDF PubMed Google Scholar). Based on the trans-membrane helices prediction using a hidden Markov model (10Sonnhammer E.L. von Heijne G. Krogh A. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998; 6: 175-182PubMed Google Scholar), there are two proposed trans-membrane helices close to the N terminus of 17β-HSD2, the first one situated in amino acids 5–27 and the second one in 34–56. The latter is much more hydrophobic than the former. The enzyme is thus suggested to be an integral membrane protein (7Puranen T.J. Kurkela R.M. Lakkakorpi J.T. Poutanen M.H. Itaranta P.V. Melis J.P. Ghosh D. Vihko R.K. Vihko P.T. Endocrinology. 1999; 140: 3334-3341Crossref PubMed Scopus (49) Google Scholar). Up to now, most of the information obtained for 17β-HSD2 is about genes and mRNA studies. Although an N-29 amino acid truncated form, in which the first proposed transmembrane helix was deleted, retained about 60% of its catalytic activity as compared with wild type in the intact cells and was purified using a detergent β-octyl glucoside, knowledge about the purification of the enzyme is still limited (7Puranen T.J. Kurkela R.M. Lakkakorpi J.T. Poutanen M.H. Itaranta P.V. Melis J.P. Ghosh D. Vihko R.K. Vihko P.T. Endocrinology. 1999; 140: 3334-3341Crossref PubMed Scopus (49) Google Scholar). In order to elucidate the structure and function of the protein, we carried out the overproduction, purification, reconstitution, and characterization of N-terminal His6-tagged full-length 17β-HSD2, which are reported here. Restriction endonucleases and modifying enzymes used in molecular biology experiments were purchased from AmershamBiosciences and Roche Molecular Biochemicals. Taq DNA polymerase and Pfu DNA polymerase were from PerkinElmer Life Sciences and Stratagene (La Jolla, CA), respectively. Spodoptera frugiperda (Sf9) cells, the Bac-N-Blue transfection kit, and the pBlue Bac4.5 transfer vector were from Invitrogen. Grace's insect cell culture medium, yeastolate, and lactalbumin hydrolysate were from Invitrogen. β-Octyl glucoside, decyl-β-d-maltoside, dodecyl-β-d-maltoside (β-DDM), polyoxyethylene 8-lauryl ether (C12E8), and Triton X-100 were from Anatrace (Maumee, OH). Radioisotopic labeled steroids were from PerkinElmer Life Sciences. His bind resin was from Novagen. SM2 Bio-Beads was from Bio-Rad. Steroids, bis-sulfosuccinimidyl suberate (BS),l-α-phosphatidylcholine (PC),l-α-phosphatidylethanolamine,l-α-phosphatidylinositol, and all other chemicals were from Sigma. The full-length human 17β-HSD2 cDNA coding sequence was obtained by PCR amplification from pCMV/17β-HSD2 (11Luu-The V. Zhang Y. Poirier D. Labrie F. J. Steroid Biochem. Mol. Biol. 1995; 55: 581-587Crossref PubMed Scopus (223) Google Scholar). A nucleotide sequence coding for 6 histidine residues followed by a Factor Xa cleavage site was added at the 5′ terminus of the 17β-HSD2 cDNA. The forward primer contained a BamHI site (underlined), 5′-CGGGATCCATGGGCAGCCATCATCATCATCATCATCATAGCAGCATCGAAGGCCGTGGCGGCATGAGCACTTTCTTCTCGGACACAGCATGG-3′, and the reverse primer contained an EcoRI site (underlined), 5′-GGAATTCTTACTAGGTGGCCTTTTTCTTGTA-3′. The 1.2-kb amplified products were digested with the appropriate enzymes and subcloned into the corresponding sites of the pBlue Bac4.5 vector. Using this method, we also constructed N-terminal His10-tagged as well as C-terminal His6-tagged 17β-HSD2 and the enzyme lacking the first 38, 52, and 61 amino acids of the N terminus (N-38-, N-52-, and N-61-deleted 17β-HSD2). The recombinant vectors were identified using dideoxynucleotide sequencing (Big DyeTM Terminator Cycle Sequencing Ready Reaction Kit, PerkinElmer Applied Biosystems, 373 sequencer with XL Upgrade). Linearized AcMNPV DNA (Bac-N-Blue DNA) (0.5 μg) was used to co-transfect monolayers of Sf9 cells in the presence of InsectinPlus liposomes, according to the manufacturer's instructions. Five days after co-transfection, the media were collected, and the recombinant baculoviruses were purified using three rounds of plaque assay in the presence of Bluo-Gal. Sf9 cells were grown as monolayers in flasks containing Grace's insect cell culture medium with 5% fetal bovine serum and maintained at 27 °C. The wild type baculovirus and the recombinant virus carrying 17β-HSD2 were used to infect the 90% confluent cells at a multiplicity of infection of 0.1–0.5 for virus amplification and an multiplicity of infection of 5–10 for protein overproduction. The infected cells were harvested 72 h postinfection, washed with cold phosphate-buffered saline, pelleted, and stored at −80 °C for later use. Cell pellets containing overexpressed N-terminal His6-tagged 17β-HSD2 were fractionated in 1.5 ml of buffer A (40 mm Tris, pH 8.0, 20% glycerol, 20 μm NAD, 0.4 mmphenylmethylsulfonyl fluoride, 0.15 m NaCl, and 1 μg/ml each of the following protease inhibitors: leupeptin, chymostatin, antipain, aprotinin, and pepstatin A) containing detergents. β-Octyl glucoside, decyl-β-d-maltoside, β-DDM, Triton X-100, sodium cholate, and C12E8 were used with concentrations 0.4–1.2%. The samples were sonicated on ice by a sonic dismembranator (Fisher Scientific) and incubated for 1 h at 4 °C. 0.1 ml of each homogenate was transferred to separate tubes as control, and the remaining homogenate was centrifuged for 45 min at 180,000 × g at 4 °C. The aliquots from the homogenates, supernatants, and pellets were analyzed by electrophoresis, immunoblotting, and activity assay. The cell pellets from 6–8 × 175-cm2flasks (which represents about 1.6–2 × 108 cells) were suspended in 50 ml of buffer A. The remaining procedures were carried out at 4 °C or on ice unless otherwise specified. The cells were lysed by sonication. The suspension was incubated for 15 min and centrifuged for 30 min at 180,000 × g. The pellets were then solubilized in 100 ml of buffer B (40 mm Tris, pH 8.0, 150 mm NaCl, 10% glycerol, 8 mmimidazole, 20 mm NAD, 0.4 mmphenylmethylsulfonyl fluoride, 0.5% β-DDM, 1 μg/ml each of the protease inhibitors) and incubated by rotating for 1 h. The supernatants were collected after centrifugation for 45 min at 180,000 × g, adjusted to 300 mm NaCl, mixed with 3 ml of nickel-chelated resin pre-equilibrated with buffer B (containing 300 mm NaCl), and incubated by rotating for 1 h. The mixture was loaded onto the column. The column was washed with 10 column volumes of buffer C (buffer B containing 300 mm NaCl, 0.3% β-DDM, 15 mm imidazole, and 15% glycerol) and 10 column volumes of buffer D (buffer B containing 45 mm imidazole, 200 mm NaCl, 0.3% β-DDM, and 20% glycerol). Bound proteins were eluted with buffer E (40 mm Tris, pH 7.5, 150 mm NaCl, 20% glycerol, 0.2% β-DDM, 250 mm imidazole, 40 μm NAD, 0.4 μm phenylmethylsulfonyl fluoride, and 1 μg/ml protease inhibitors). The fractions with high 17β-HSD2 activity were collected, frozen in liquid nitrogen, and stored at −80 °C. The purified enzyme without adding cofactor NAD in the purification procedures was used to detect cofactor kinetic constants. The reconstitution method basically depended on the strategies described by Rigaud (12Rigaud J. Pitard B. Levy D. Biochim. Biophys. Acta. 1995; 1231: 223-246Crossref PubMed Scopus (405) Google Scholar). Three kinds of phospholipid (PC, phosphatidylethanolamine, and l-α-phosphatidylinositol) were tested in the reconstitution system. They were mixed with different ratios, which were around the lipid compositions of the human liver. The mixtures were dissolved in chloroform and dried under a stream of nitrogen gas to minimize the lipid oxidation. The remaining trace of solvent was removed under vacuum for at least 2 h. The dried films were suspended in buffer F (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, and 0.5 mm DTT) at a concentration of 20 mg of lipids/ml. The liposomes were obtained by sonication (1-s burst, 5-s interval for 45 min at output 2.0) in an ice bath under nitrogen gas. The suspensions were frozen in liquid nitrogen and thawed at room temperature three times. The liposomes were extruded through 400-nm polycarbonate membranes (Nuclepore® Track-Etch Membrane; Corning) three times, frozen in liquid nitrogen, and stored at −80 °C. To determine the physical state of the detergent-solubilized liposomes, the liposomes were aliquoted and mixed with different amounts of β-DDM in a final concentration of 4 mg of lipids/ml using buffer F. The samples were incubated at room temperature for 3 h under constant agitation. The turbidity of the different phospholipid-detergent suspensions was measured at 540 nm with a spectrophotometer (Backman DU 7400). To reconstitute 17β-HSD2, the liposomes were diluted to 4 mg of lipids/ml, saturated with β-DDM, and equilibrated under constant agitation for 3 h at room temperature. The purified N-terminal His6-tagged 17β-HSD2 was adjusted to 0.15 mNaCl and the same concentration of detergent as that in the saturated liposomes, mixed with the saturated liposomes in a ratio of liposomes to protein of 14:1 (w/w), and incubated for 2 h at 4 °C under rotating. The detergent was removed by three successive extractions with 80 mg/ml, wet weight, polystyrene beads and rotated at 4 °C. The first, second, and third extractions lasted for 2, 2, and 4 h, respectively. The beads were removed by filtration over glass wool. Then the mixture was adjusted to 6% glycerol using buffer F. The proteoliposomes were harvested by centrifugation at 180,000 ×g for 45 min, resuspended in a buffer (40 mmTris, pH 7.4, 20% glycerol, 150 mm NaCl, 1 mmEDTA, and 0.5 mm DTT), and stored at −80 °C. The activity of 17β-HSD2 was monitored by the spectrophotometric assay. It was initiated by the addition of 17β-HSD2 in 0.5 ml of reaction mixture (50 mmsodium carbonate, pH 9.2, 1 mm NAD, and 25 μmtestosterone). The reactions were monitored by spectrophotometric measurement of the reduction of NAD at 340 nm. A reaction mixture containing no cofactor or substrate was used as control. One unit of enzyme activity is defined as 1 μmol of product formed in 1 min. Enzyme activities in cultured cells were measured by plating the cells in the six-well plates at a density of 1.2 × 106/well. The cells were set for 1 h to attach followed by the infection of virus at a multiplicity of infection of 10. A mock infection was set as a background control. After a 50-h incubation at 27 °C, the medium was removed, and 2 ml of serum-free TNM-FH medium with 10 μmof each 14C-labeled estrone, estradiol, testosterone, androstenedione (4-dione), and dihydrotestosterone was added to each well. The reaction was set at room temperature, at different time intervals (3, 10, 20, 40, and 60 min), and aliquots of the media were moved to the tubes containing cold diethyl ether. The steroids were extracted and quantified as mentioned under “Steady-state Kinetics.” The kinetic constants of 17β-HSD2 were determined using purified and reconstituted N-terminal His6-tagged 17β-HSD2. The reaction mixture contained 50 mm sodium phosphate buffer, pH 7.4, 1 mmconstant concentration of cofactor NAD, with different steroid substrates ([14C]estradiol, [14C]testosterone, [14C]dihydrotestosterone, and [3H]20α-dihydroxyprogesterone), and concentrations varied from 0.14 to 4 μm for the kinetic constants of steroids. The same buffer system contained a 10 μmconstant concentration of steroids (estradiol and testosterone) with various concentrations of cofactor NAD (0.05–1 mm) for the kinetic constants of NAD. The same buffer with 10 μm of constant concentration of 4-dione and estrone and with various concentrations of cofactor NADH (0.001–0.2 mm) was used for the kinetic constants of NADH. The kinetic constants of NADP were determined with a 10 μm constant concentration of estradiol and various concentrations of cofactor NADP (0.6–10 mm). The initial velocity was measured with less than 5% substrate conversion. The reactions were carried out at 37 °C and stopped by removing 0.5 ml of reaction mixture to the cold diethyl ether at four different time intervals (0, 20, 40, and 60 s). The steroids were extracted with ethanol in dry ice and dried by evaporation. They were then dissolved in dichloromethane, applied onto thin layer chromatograms (TLC), separated by toluene/acetone (4:1, v/v), and quantified by Storm 860 Laser Scanner (Molecular Dynamics, Inc., Sunnyvale, CA; ImageQuant software). At least three independent experiments were carried out for each kinetic constant. The kinetic results were fitted for the Michaelis-Menten equation and calculated using a Lineweaver-Burk plot. The values of the catalytic constant,k cat, were calculated from theV max values with the homodimer molecular mass of 90 kDa (k cat is the turnover number,i.e. the number of moles of substrate transformed per second per mole of enzyme). The apparent functional molecular mass of N-terminal His6-tagged 17β-HSD2 was estimated by cosedimentation with protein standards on 8–30% glycerol gradients. Glycerol gradients (13 ml; Beckman SW 40Ti rotor) were prepared by using a gradient maker with equal volumes of 8 and 30% glycerol buffer containing 20 mm Tris, pH 7.4, 0.15 m NaCl, 40 μm NAD, 1 mm EDTA, 0.5 mm DTT, and 0.1% Triton X-100. The glycerol gradients were equilibrated at 4 °C for about 8 h before loading the samples. The samples contained 20 μg of purified and reconstituted 17β-HSD2, 100 μg of each protein standard (rabbit skeletal muscle aldolase, bovine serum albumin, and chicken ovalbumin (13Sigma Technical Bulletin No. MKR-137. St. Louis, MO1986Google Scholar)), and the same buffer as in the gradient, but the glycerol concentration was less than 8%. The samples were equilibrated at 4 °C for 1 h and then layered on top of the glycerol gradients and centrifuged at 40,000 rpm for 40 h at 4 °C. The gradients were fractionated from the bottom into 0.3-ml fractions. 17β-HSD2 fractions were verified by the enzyme activity assay and Western blot. The positions of the standard markers were determined by SDS-PAGE. This was performed according to the method described by Knoller (14Knoller S. Shpungin S. Pick E. J. Biol. Chem. 1991; 266: 2795-2804Abstract Full Text PDF PubMed Google Scholar) with modifications. 60 μl of reaction buffer contains 50 mm potassium phosphate, pH 7.4, 20% glycerol, 1 mm EDTA, 0.5 mm DTT, 3 μg of purified and reconstituted N-terminal His6-tagged 17β-HSD2, and cross-linking reagent BS with concentrations at 0, 0.25, 1, and 3 mm, respectively. The reaction proceeded for 30 min at 25 °C and stopped by adding glycine to a final concentration of 30 mm. The samples were analyzed by 5–15% gradient SDS-PAGE followed by Western blot. SDS-PAGE was performed according to the method of Laemmli (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) using 12% polyacrylamide gel or 5–15% gradient SDS-PAGE. The samples in reducing loading buffer were incubated at 40 °C for 30 min instead of boiling before loading to prevent the aggregation of the membrane protein (16Schagger H. von Jagow G. Schagger H. A Practical Guide to Membrane Protein Purification. Academic Press, Inc., San Diego, CA1994: 59-79Crossref Google Scholar). The gel after migration was stained with Coomassie Blue. For Western blot analysis, blots were probed with rabbit polyclonal antibody raised against human 17β-HSD2 as the first antibody and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit polyclonal antibody (AmershamBiosciences) as the second antibody. The immunoreactive blots were detected with ECL reagents (PerkinElmer Life Sciences) and exposed to x-ray film (Eastman Kodak Co.). Protein concentrations without detergent were determined using the Bradford reagent (Bio-Rad). The concentrations of proteins with detergents or with phospholipid were determined by the method of microgram quantities of protein determination (17Kaplan R.S. Pedersen P.L. Anal. Biochem. 1985; 150: 97-104Crossref PubMed Scopus (187) Google Scholar) to prevent the alteration of detergents and phospholipids in the protein concentration by conventional methods. Human 17β-HSD2 cDNA with a 6-histidine coding sequence and a Factor Xa cleavage site at its 5′ terminus was subcloned into the baculovirus transfer vector pBlueBac 4.5. The incorporation of the Factor Xa cleavage site allowed the removal of the His tag after purification of the recombinant protein, leaving only two additional glycines at the N terminus of 17β-HSD2. Sf9 cells were co-transfected with Bac-N-Blue DNA and the above transfer vector of 17β-HSD2 to produce the recombinant baculovirus. Protein expression was optimized by evaluating the expression levels of the infection at different time intervals. The activity was first detected 24 h postinfection and reached a maximum between 60 and 72 h postinfection (Fig.1, A and B), whereas no activity could be detected in wild-type AcMNPV virus-infected cells. Thus, the protein expression conditions were set as follows: infection of the cells at a multiplicity of infection from 5 to 10 and harvest in 72 h postinfection. Under these conditions, the overexpressed 17β-HSD2 constitutes about 3% of the total protein in the insect cell lysate, with a specific activity of 0.012 units/mg in the cell homogenate. Using the same method, we also overproduced N-38-, N-52-, and N-61-deleted 17β-HSD2, as well as N-terminal His10-tagged and C-terminal His6-tagged 17β-HSD2. The truncated N-38 form was expressed at about 2% of the total protein in the insect cell lysate, with a specific activity of 0.005 units/mg in the cell homogenate. Although this form could be solubilized to a higher level with detergent from membrane vesicles (solubilized 45% of the enzyme in the presence of 0.4% β-DDM) than that of N-terminal His6-tagged 17β-HSD2 (solubilized 30.5% of the enzyme in the presence of 0.4% β-DDM), it was unstable in the solubilized state with detergents even in intact cells and showed a very strong tendency to degrade and aggregate in the cell homogenate. The truncated N-52 and N-61 forms were expressed in fairly low amounts in Sf9 insect cells. The N-52 form was even more unstable than the N-38 form. We found that the N-52 form in fresh cultured cells still retained a little activity, but it completely lost activity in several hours at 4 °C. Moreover, the N-61 form was totally inactive in fresh cultured cells. The N-terminal His10-tagged 17β-HSD2 was expressed to a high level (about 5% of the total protein); however, it retained a quite lower specific activity (about 0.001 units/mg) than that of the N-terminal His6-tagged form (about 0.012 units/mg). The C-terminal His6-tagged 17β-HSD2 was expressed at about 2% of the total protein in the insect cell lysate, with a specific activity of 0.009 units/mg in the cell homogenate. This recombinant was solubilized to a lower level from membrane vesicles (solubilized 21% of the enzyme in the presence of 0.4% β-DDM) than that of N-terminal His6-tagged 17β-HSD2 and exhibited a very high tendency to aggregate, as seen in SDS gel and Western blot analysis. Indeed, the majority of the protein presented as a polymer staying in the sample-loading place or as a dimer (data not shown). These findings suggest that there is a stronger membrane interaction in C-terminal His6-tagged 17β-HSD2 than in N-terminal His6-tagged 17β-HSD2. Furthermore, several purification tests using various detergents demonstrated that the C-terminal His6 tag of this form was not able to be effectively bound to nickel-chelated affinity matrix. Finally, N-terminal His6-tagged 17β-HSD2, although still highly labile, was found to be able to retain full biological activity, to be expressed in a fairly good amount in the baculovirus expression system, and to facilitate its purification. Therefore, this form was chosen in our study. N-terminal His6-tagged 17β-HSD2 is still a very labile protein with a strong tendency to aggregate and degrade. The choice of an optimal detergent is the crucial step in the purification. Much effort was devoted to finding a suitable detergent to solubilize this recombinant from the cell membranes. Several commonly used detergents were tested for the protein stability, solubility, and binding capacity with nickel matrix. The results are summarized in Table I. The pH during solubilization was kept at 8.0, since the protein was subsequently used to bind to the Ni2+-agarose matrix. Sodium cholate showed no significant inhibition of 17β-HSD2 activity, but the protein solubility was very low with this detergent. β-Octyl glucoside had low ability in protein solubilization and strong inhibition to the enzyme activity at increasing concentrations. C12E8 and decyl-β-d-maltoside displayed medium ability both in solubilizing and in maintaining the enzyme activity. Triton X-100 showed high protein solubility, but it significantly inhibited the enzyme activity. Although none of the detergent could assist the enzyme to get more than 50% solubility, β-DDM gave the best results both in solubilizing and in maintaining the enzyme activity among those detergents tested. Typically, we used 0.5% β-DDM in the solubilization of His6-tagged 17β-HSD2, considering that higher concentrations of β-DDM did not further improve the solubility notably but rather inhibited the enzyme activity and solubilized more contaminants.Table IDetergent solubility testDetergentsDetergent concentrationSolubilityActivity in homogenate % % units/mlNo detergent0.40.014Sodium cholate0.40.60.0140.81.30.0141.22.30.015β-octyl glucoside0.43.80.0150.813.40.0111.2160.006C12E80.411.80.0120.814.50.0121.221.80.010Triton X-1000.432.90.0090.836.20.0061.238.90.004Decyl-β-d-maltoside0.425.10.0160.827.60.0" @default.
• W1988205364 created "2016-06-24" @default.
• W1988205364 creator A5048528146 @default.
• W1988205364 creator A5079671959 @default.
• W1988205364 creator A5080203487 @default.
• W1988205364 date "2002-06-01" @default.
• W1988205364 modified "2023-10-16" @default.
• W1988205364 title "Purification, Reconstitution, and Steady-state Kinetics of the Trans-membrane 17β-Hydroxysteroid Dehydrogenase 2" @default.
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