Purification, Reconstitution, and Steady-state Kinetics of the Trans-membrane 17β-Hydroxysteroid Dehydrogenase 2

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 in which the first proposed was retained about of its catalytic activity as with type in the and was purified using a detergent about the purification of the enzyme is (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 to the structure and of the protein, carried out the purification, reconstitution, and of 17β-HSD2, which are reported and enzymes in molecular were from and and were from and the and the were from insect cell and were from dodecyl-β-d-maltoside and were from were from His was from was from bis-sulfosuccinimidyl suberate and were from The human 17β-HSD2 cDNA was obtained by from V. Y. D. J. Biochem. Mol. Biol. PubMed Scopus Google Scholar). for 6 followed by a was at the terminus of the 17β-HSD2 The a and the an The were with the enzymes and into the of the Using this also as well as 17β-HSD2 and the enzyme the first and amino acids of the N terminus and The recombinant were using with was to of Sf9 in the of to the the were and the recombinant were purified using of in the of Sf9 were as in insect cell with and at The type and the recombinant 17β-HSD2 were to the 90% at a of of for and an of of for protein The were with and at for overexpressed 17β-HSD2 were in of μm m and of the and and were with concentrations The were on by a and for at of was to as and the was for at at The from the and were by and activity The cell from about were in of A. The were carried out at on The were by The was for and for at The were in 100 of of the and by for The were for at to with of with and by for The was onto the The was with 10 of and and 10 of and were with μm μm and The with high 17β-HSD2 activity were in and at The purified enzyme NAD in the purification was to kinetic The reconstitution on the by J. D. PubMed Scopus Google Scholar). of and were in the reconstitution were with which were the of the human The were in and a of to the The of was removed for at 2 The were in and at a of of The liposomes were obtained by for at in an The were in and at The liposomes were in and at the of the detergent-solubilized the liposomes were and with amounts of in a of of using The were at for The of the was at with a 17β-HSD2, the liposomes were to of with and for at The purified 17β-HSD2 was to and the of detergent as that in the with the liposomes in a of liposomes to protein of and for 2 at The detergent was removed by with polystyrene and at The and for and The were removed by the was to glycerol using The were by at for in a and and at The activity of 17β-HSD2 was by the It was by the of 17β-HSD2 in of and The were by of the of NAD at was as of enzyme activity is as of in in were by the in the at a of 1.2 The were for to followed by the of at a of of was as a a at the was and 2 of with 10 estradiol, testosterone, androstenedione and dihydrotestosterone was to The was at at and and of the were to the The were and as The kinetic constants of 17β-HSD2 were using purified and reconstituted The of with steroid substrates and and concentrations from to μm for the kinetic constants of The a 10 of and with concentrations of NAD for the kinetic constants of The with 10 μm of of and and with concentrations of was for the kinetic constants of The kinetic constants of NADP were with a 10 μm of estradiol and concentrations of NADP The was with than The were carried out at and by of to the at and The were with in and by were in onto by and by were carried out for kinetic The kinetic were for the and calculated using a The of the catalytic were calculated from with the molecular mass of kDa is the the of of second of The functional molecular mass of 17β-HSD2 was by with protein on glycerol were prepared by using a gradient with of and glycerol m μm and The glycerol were at for about the The of purified and reconstituted 17β-HSD2, 100 of protein and and the as in the the glycerol was than The were at for and on of the glycerol and at for at The were from the into 17β-HSD2 were by the enzyme activity and The of the were by This was to the by S. S. J. Biol. Chem. Full Text PDF PubMed Google with of of purified and reconstituted 17β-HSD2, and cross-linking with concentrations at and The for at and by to a of The were by gradient followed by was to the of Nature. PubMed Scopus Google using gradient The in reducing were at for of to the of the membrane protein von G. to Google Scholar). The was with For were with human 17β-HSD2 as the first and as the second The were detected with and to concentrations detergent were using the The concentrations of with with were by the of of protein Biochem. PubMed Scopus Google to the of and in the protein by Human 17β-HSD2 cDNA with a and a at its terminus was into the The of the the of the His purification of the recombinant protein, two at the N terminus of Sf9 were with and the of 17β-HSD2 to the recombinant expression was by the expression of the at The activity was first detected and a between and and activity be detected in cells. the protein expression were as of the at a of from to 10 and in the overexpressed 17β-HSD2 about of the protein in the insect cell with a activity of in the cell Using the also and 17β-HSD2, as well as and The truncated form was expressed at about of the protein in the insect cell with a activity of in the cell Although this form be to a higher with detergent from membrane of the enzyme in the of than that of 17β-HSD2 of the enzyme in the of it was in the with in and a to and in the cell The truncated and forms were expressed in amounts in Sf9 insect cells. The form was more than the found that the form in retained a activity, it activity in at the form was inactive in cells. The 17β-HSD2 was expressed to a high of the it retained a activity than that of the form The 17β-HSD2 was expressed at about of the protein in the insect cell with a activity of in the cell This recombinant was to a from membrane of the enzyme in the of than that of 17β-HSD2 and a high to as in and the of the protein as a in the as a that there is a membrane in 17β-HSD2 than in Furthermore, purification using demonstrated that the of this form was able to be to 17β-HSD2, highly was found to be able to biological activity, to be expressed in a in the expression and to its this form was in our study. 17β-HSD2 is a protein with a to and The of an detergent is the crucial in the was to a suitable detergent to this recombinant from the cell were for the protein and with The are in The was at the protein was to to the of 17β-HSD2 activity, the protein was with this had in protein and to the enzyme activity at and both in and in the enzyme high protein it the enzyme Although of the detergent the enzyme to more than the best both in and in the enzyme activity in the of 17β-HSD2, that higher concentrations of the the enzyme activity and more in in a The purification was carried out in a using as The are in and are in and were removed by the with and 17β-HSD2 was purified with a purity of more than 90% based on and were two with molecular of about and kDa on the which were to be a form and a by The of the purification was about of protein with a activity of about using estradiol as from 2 found that the enzyme activity in both the and the using as detergent be for at the and purified 17β-HSD2 had a to that the protein at for and and activity, The of the had to be by as as and at for of 17β-HSD2 overexpressed in Sf9 in a from the of and from were to their to 17β-HSD2 The from demonstrated the activity and were thus for the protein the of the the at was at concentrations of 17β-HSD2 was for the into of liposomes with the liposomes the of the and The concentrations of at to and The of the liposomes with and the of the reconstituted 17β-HSD2 are in The activity was obtained the liposomes with the liposomes with a were in the reconstitution The in the on the of the liposomes at the of the reconstitution also on the the of and the were and the was Using a in the reconstitution the enzyme was able to into the liposomes and using a higher the were active The of glycerol at than in protein and detergent to of enzyme the of reconstitution, the were by the glycerol was to to the to an of glycerol the to be It was also found that using high in the reconstitution a higher of active than that in a The reconstituted 17β-HSD2 using our higher activity (2.6 with estradiol) and much higher stability than The were at for 2 of enzyme The reconstituted protein was also than to the of reconstitution The mass of this protein was calculated based on the of protein and the 17β-HSD2 on the reducing and kDa was obtained in the of the His about 2 kDa for the amino This calculated molecular mass well with the molecular mass of 42,782 predicted from the amino acid (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). The functional molecular mass of this protein was calculated based on glycerol gradient with which a molecular mass of 90.4 ± 1.2 kDa The that the protein was present in the to the of 17β-HSD2 17β-HSD2 were detected in the of the the of the a cross-linking using as was one at and one at in the control In the with and of the with the were of and The form of the protein in the the that 17β-HSD2 as a in the cross-linking of and 17β-HSD2 were with concentrations of were by gradient and were by protein with and The kinetic constants for substrates estradiol, dihydrotestosterone, and and for and NADP are in The for steroid substrates were close to in cell (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 and in purified 17β-HSD2 (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). also the kinetic constants for with concentrations of testosterone, and and NADP with of to the for substrates, the for NAD between two oxidative substrates and and the for between two substrates and were also Although the for NAD were higher and the were also higher than for the enzyme had the catalytic for both oxidation and it was in of oxidative in Sf9 and the were reported in (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, V. Y. D. J. Biochem. Mol. Biol. PubMed Scopus Google Scholar). The for NADP with the estradiol as at a 9600 more than higher than that for NAD with the substrate. This suggests that the in the is a to the It is well that the as and the dehydrogenase NAD as in a of the kinetic and the of NAD is higher than that of with a of about D. Scholar), the be in the oxidation by using NAD as for this enzyme in constants of substrates in the oxidation with purified reconstituted His 6 K μm m ± ± ± ± ± ± ± ± K and ± of were calculated from with the molecular mass of in a constants of with purified reconstituted His 6 K μm m ± ± ± ± ± ± ± ± ± ± K m and ± of were calculated from with the molecular mass of in a The K and ± of were calculated from with the molecular mass of The K m and ± of were calculated from with the molecular mass of in the there are two proposed trans-membrane helices at the N terminus of 17β-HSD2, the more hydrophobic on the second proposed trans-membrane have to a truncated which was able to enzyme activity as well as found that the truncated form retained about of its catalytic activity as with the this form was in the cell in the detergent-solubilized For the the form retained activity in and the form was It was reported that the truncated N-29 17β-HSD2 which the first proposed trans-membrane was retained about of its catalytic activity as with the type enzyme (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). the truncated form was inactive (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). of that the first proposed trans-membrane is important and that the second one is crucial in the enzyme found that 17β-HSD2 was the most suitable form to our overexpressed 17β-HSD2 demonstrated that 17β-HSD2 was much more hydrophobic than the This suggests that the His on the N terminus its purification also the on the of the enzyme. the protein be hydrophobic than the one His the protein retained about of the type catalytic activity, and this that the His may the enzyme also found that the of the form was higher than the form and than the This suggests that close to the N terminus of the protein a higher of the to the primary structure of human 17β-HSD2, there is a hydrophobic core possessing 33 nonpolar amino acids close to its N terminus and a motif in the (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), which that 17β-HSD2 has a high to in the detergent-solubilized Based on this had been in the purification and reconstitution an of glycerol was found that glycerol an important role in the protein, high of glycerol the of the 17β-HSD2 with glycerol was in the the protein with and glycerol was in the found that the the detergent on the hydrophobic of the protein, that it the the of the protein with excessive high m enzyme and of m was thus the protein with and a of was in the the of and was amounts of the and for Using a large of and large of using a on the to the protein and to its found that NAD had an on the enzyme. Using NAD in the purification to an enzyme with higher The purified 17β-HSD2 was to its activity, and the protein be by m and than by a widely to between and integral membrane of Scholar). Based on of 17β-HSD2 to be an integral membrane active 17β-HSD2 for biological and to to the enzyme. reconstituted the enzyme using the The purified and 17β-HSD2 was to the enzyme The reconstituted protein has the physiological as reported (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). The with is with the obtained using 17β-HSD2 (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). The with substrates in the oxidation are to the from 17β-HSD2 cell (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 and from purified 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). the for substrates in the oxidation are higher than of demonstrated that the reconstituted protein is of the that the for NAD is higher than that for and both have the kinetic 17β-HSD2 is a dehydrogenase in The for NAD and are also reported in its with cell D. Y. J. Endocrinol. 1997; PubMed Scopus Google Scholar), which are μm for NAD and μm for with and as substrates, the for the enzyme were from the the of the cell is it is well that of NAD is higher than that of and is higher than that of NADP D. Scholar). It is also well that the use as and the use NAD as in V. Y. D. J. Biochem. Mol. Biol. PubMed Scopus Google Scholar). This that the in the is a to the that the on oxidation using NAD as glycerol gradient in the of concentrations of was to the functional molecular mass of found that the of in the of the gradient in the of and this is with the gradient ultracentrifugation of reconstituted membrane protein P. PubMed Scopus Google Scholar). of in the the enzyme activity was from to more than its functional molecular mass was to be in the in this that 17β-HSD2 was present in the to the of 17β-HSD2 activity the activity was high in the that the enzyme retained activity in higher concentrations of the together with L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google that with protein functional molecular mass in glycerol The enzyme with it retained much higher activity than with the detergent molecular mass to in the in the glycerol Using higher than in the the enzyme its This demonstrated that the detergent with the protein functional molecular mass in the glycerol In this an to highly integral membrane 17β-HSD2 enzyme. The overexpressed 17β-HSD2 was demonstrated to be the most suitable form in our study. is the best detergent in both the protein and the enzyme in an active 17β-HSD2 was proved to be a with a molecular mass of 90.4 ± 1.2 kDa in the of a 2-kDa His purification and reconstitution a and to and reconstituted This to the cell and recombinant protein protein to and structure studies. The have may be for membrane steroid for in this also V. for the and the for 17β-HSD2 and M. for the of the

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