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Purification of full-length recombinant human and rat type 1 11β-hydroxysteroid dehydrogenases with retained oxidoreductase activities
Protein Expression and Purification 26 (2002) 349–356 www.academicpress.com
Purification of full-length recombinant human and rat type 1 11b-hydroxysteroid dehydrogenases with retained oxidoreductase activities C. Stefan I. Nobel,a,b Finn Dun as,a,1 and Lars. B. Abrahmsena,* a b
Biovitrum AB, Division of Pharmaceuticals, Department of Assay Development and Screening, S-112 87 Stockholm, Sweden Unit for Biochemical Toxicology, Department of Biochemistry and Biophysics, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden Received 6 February 2002, and in revised form 8 July 2002
Abstract 11b-Hydroxysteroid dehydrogenase type 1 (11b-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum. This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase. Previous attempts to purify the native enzyme have yielded a protein without reductase activity. To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11b-HSD1 with retention of their natural oxidoreductase activities. This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography. Both enzymes were 90–95% pure and exhibited dehydrogenase and reductase activities with KM values in agreement with those reported in the literature. Ó 2002 Elsevier Science (USA). All rights reserved.
11b-Hydroxysteroid dehydrogenase type 1 (11bHSD1) plays a key role in regulating local tissue glucocorticoid concentrations by converting cortisone into the active glucocorticoid cortisol in humans and 11-dehydrocorticosterone into corticosterone in rodents (reviewed in [1]). Gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels and for the up-regulation of key hepatic gluconeogenic enzymes. This enzyme is expressed primarily in the liver, adipose tissue, brain, gonads, and blood vessels [1]. The phenotype of transgenic mice in which 11b-HSD1 is over-expressed in adipose tissue indicates that this enzyme plays a role in connection with syndrome X and diabetes [2]. Native 11b-HSD1 is a member of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins *
Corresponding author. E-mail address: [email protected] (L.B. Abrahmsen). 1 Present address: Affibody AB, P.O. Box 20137, S-16102 Bromma, Sweden.
(reviewed in [3]). This 34 kDa, glycosylated, NADP(H)dependent enzyme is attached to the endoplasmic reticular membrane and faces the lumenal compartment. The enzyme is bidirectional in vitro, but is believed to predominantly function as an oxidoreductase in vivo [1]. Surprisingly, the oxidoreductase activity of 11b-HSD1 is more sensitive to inactivation than is the dehydrogenase activity, both in cell extracts and following purification of the enzyme [4,5]. This unexpected property prompted us to investigate the catalytic mechanism of 11b-HSD1 in greater detail. To enable such studies, we decided to purify the recombinant enzyme. Recombinant fulllength 11b-HSD1 has not been purified previously and when purified from natural sources only dehydrogenase activity has been retained. Materials and methods Materials Chemicals and reagents were purchased from Sigma– Aldrich (Stockholm, Sweden) or Merck Eurolab
1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 5 9 2 8 ( 0 2 ) 0 0 5 4 7 - 8
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(Stockholm, Sweden), if not otherwise stated. [1,23 H(N)]-Hydrocortisone, 52 Ci/mmol, was obtained from NEN Life Sciences (Zaventem, Belgium), while [1,2,6 7-(3 H)]-corticosterone, 77 Ci/mmol, and the customsynthesized [1,2(n)-3 H]-cortisone, 46 Ci/mmol, and 11dehydro-[3 H]-corticosterone, 61 Ci/mmol, were supplied by Amersham Pharmacia Biotech (Amersham, UK). Oligonucleotides were custom-made by Life Technologies (Paisley, Scotland) and DNA plasmid purification kits were purchased from Qiagen (Hilden, Germany). Cloning of 11b-HSD1 and generation of yeast clones expressing this enzyme The human 11b-HSD1 gene was cloned from HepG2 cells by RT-PCR and found to encode a protein with a primary sequence identical to the published one [6]. An N-terminal 6His tag containing a cleavage site for picornavirus protease 3C [7] was fused to the hydrophobic anchoring domain of the enzyme by employing two consecutive PCRs (using the Expand High Fidelity PCR system from Roche Diagnostics, Bromma, Sweden, according to supplierÕs protocol and with an annealing temperature of 55 °C). The primers used for the first amplification were forward 1: 50 -TTA GAA GCT TTA TTT CAA GGG CCA ATG GCT TTT ATG AAA AAA TAT CTC CTC CCC ATT CTG GG-30 and reverse 1: 50 -GCG CGG CCG CCT ACT TGT TTA TGA ATC TGT CCA TAT TAT AGC TCG-30 ); while the primers for the second reaction were forward 2: 50 -CGG AAT TCG GTA CC TAC AAA ATG TCT CAC CAT CAC CAT CAC CAT TTA GAA GCT TTA TTT CAA GGG CC-30 and reverse 1 (see above). The final PCR product coded for human 11b-HSD1, preceded by the amino acid sequence MSHHHHHHLE ALFQGP (Fig. 1) and contains sites for EcoRI and KpnI as well as a yeast optimal translation start sequence TACAAAATG at the 50 -end [8]. The reverse primer introduced a NotI site after the stop codon. This construct was inserted between EcoRI and NotI in the vector pPIC3.5K (Invitrogen, Groningen, The Netherlands), to obtain pMB1250. This recombinant plasmid DNA was linearized with PmeI and transformed into the Pichia pastoris strain GS115 according to supplierÕs protocol (Invitrogen). Rat 11bHSD-1 was obtained by RT-PCR employing total RNA from Sprague–Dawley rat liver (kit from Perkin–Elmer, Applied Biosystems, Stockholm, Sweden; with an annealing temperature of 55 °C). The primers (forward 3: 50 -CG GAA TTC GGT ACC GCC GCC ACC ATG AAA AAA TAC CTC CTC CCC G-30 and reverse 2: 50 -GC TCT AGA GCG GCC GC GTT GCT TAC AAA TAG GTC CCT G-30 ) added sites for EcoRI, KpnI, and a Kozak sequence at the 50 -end, and removed the stop codon and added NotI and XbaI sites at the 30 end. The PCR product was inserted into pPICZaA (In-
Fig. 1. Updated rat 11b-HSD1 sequence and sequence of constructs used for purification. (A) Amino acid sequence alignment of published 11b-HSD1 orthologs. The conserved motif (KEECALEIIKG) near the C-terminal is not contained in the published rat 11b-HSD1 sequence (deviations towards other 11b-HSDs highlighted), but is found in the one reported here. (Squirrel monkey is abbreviated Sq. monkey). (B) The N-terminal modification of both rat and human 11b-HSD1 including the 6His sequence and the Picornavirus 3C protease site (underlined). Sequence in bold represents the amino-terminal portion of human 11b-HSD1.
vitrogen) employing EcoRI and NotI to yield pMB1020. This plasmid adds the yeast secretory a-factor at the Nterminus plus a myc epitope and a 6His tag at the C-terminus of rat 11b-HSD1. Six of the clones were sequenced (cycle sequencing using BigDye Terminator Kit from Applied Biosystems) and compared to the published rat 11b-HSD1 DNA sequence [9] (see text for details). The plasmid pMB1020 was the template in a subsequent PCR (Expand High Fidelity PCR system from Roche Diagnostics) performed at an annealing temperature of 55 °C with a new set of primers (forward 4: 50 CG GAA TTC TAC AAA ATG TCT CAC CAT CAC CAT CAC CAT TTA GAA GCT TTA TTT CAA GGG CCA ATG AAA AAA TAC C-30 , reverse 3: 50 GC GCG GCC GCT CAG TTG CTT ACA AAT AGG TCC CTG-30 ). This introduced the same modification of the protein as in the case of human 11bHSD-1 (see above). The PCR product was inserted between EcoRI and NotI in pPICZB (Invitrogen) to yield pMB1116. This plasmid was linearized with PmeI and transformed into the P. pastoris strain XL33 (Invitrogen) using Zeocin as the selecting agent for integration into the genome according to supplierÕs protocol (Invitrogen). Expression in yeast The recombinant P. pastoris strains GS115 and XL33 containing genes encoding N-terminal 6His (N-His)-
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tagged human 11b-HSD1 and rat 11b-HSD1, respectively, exhibited Mutþ (methanol using plus) phenotypes. One colony from a fresh agar plate (grown in minimal dextrose medium (MD) agar plate in the case of the former strain and in yeast extract peptone containing Zeocin in the case of the latter strain), was transferred into 50 mL BMGY and incubated at 30 °C for 15 h in a 300-mL Tunair flask with shaking (Shelton Scientific, Shelton, CT). BMGY contains 1% yeast extract (Merck), 2% peptone (Difco, Detroit, MI), 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base (YNB), 1% glycerol, and 4 105 % biotin. The culture was then transferred into 200 mL fresh BMGY in a 2.5-L Tunair flask and incubated for an additional 6 h with shaking. A fermentor (3-L working volume, Belach Bioteknik AB, Sweden) containing the components of BMGY (but with less phosphate buffer, i.e., 42 mM, and more glycerol, (4%)) and 0.5 mL/L of an antifoaming agent (Adekanol) was in situ sterilized. Filter-sterilized YNB and biotin were subsequently added. A probe connected to a methanol sensor (Raven Biotech, Canada) was then aseptically mounted onto the fermentor. The fermentor was aerated with 1 vvm air and the dissolved oxygen tension (DOT) was maintained at 40% atmospheric pressure by mixing. In some cases, a 1:1 air/oxygen mixture was used towards the end of the growth period to achieve a sufficiently high DOT. The temperature was set at 30 °C and the pH was maintained at 6 by automatic addition of 25% ammonia when required. Addition of 50% glycerol (20–30 g/L h, containing 1.2% PTM1 trace element solution; Invitrogen) was initiated when the original glycerol had been depleted (as indicated by a peak signal in the DOT). After a few hours of such addition, glycerol was replaced by methanol containing 1.2% PTM1, which was pumped manually into the fermentor until a concentration of 0.2% (v/v) had been reached. The methanol sensor was used to maintain the methanol concentration at same level throughout cultivation. The cultures were harvested approximately 16–24 h after the initial addition of methanol, at which point the OD600 was 60–90. One hundred grams cell aliquots were dissolved in 100 mL buffer (20 mM NaH2 PO4 , pH 7, with 15% glycerol) and frozen at )20 °C until use for the preparation of microsomes. Preparation of microsomes Suspensions containing 100 g cells were diluted to 0.3 g/mL with buffer, giving a final concentration of 20 mM NaH2 PO4 , pH 7, and 9% glycerol as well as 1:5 complete protease inhibitor cocktail (Roche Diagnostics). Microsomes were produced by processing this cell suspension in a BioNeb Cell disintegrator (Glas-Col, Terre Haute, IN), under nitrogen gas at a pressure of 11
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bars (60–90 min per 167 mL (50 g) cells in continuous recycling mode). In pilot experiments, yeast cells were disrupted employing the zymolyase procedure to obtain spheroplasts [10]. The cell homogenate was centrifuged at 9000g for 15 min and the pellet was discarded. The resulting supernatant was centrifuged at 140,000g for 60 min and the pellet (microsomes) resuspended in buffer (40 mM NaH2 PO4 , pH 7.5, 500 mM NaCl, 10% glycerol, and 1 complete protease cocktail) using a homogenizer with a Teflon pestle and then frozen in aliquots at )70 °C. Microsomal protein content was determined using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA) or the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL). Purification of 11b-HSD1 Frozen microsomes were thawed on ice and diluted to 10 mg/mL in TPSG extraction buffer (containing 40 mM NaH2 PO4 , pH 7.5, 500 mM NaCl, 5% glycerol, 1% Triton X-100, and 1 complete protease inhibitor cocktail). Microsomal protein was solubilized by gently mixing this suspension for 30 min at 4–8 °C. The mixture was then centrifuged at 105,000g for 60 min and the pellet was discarded. Subsequently, chromatography was performed using a TALON cobalt ion-based resin (BD Biosciences Clontech, Palo Alto, CA), according to manufacturerÕs instructions. Briefly, the solubilized microsomal fraction (10 mL) was allowed to bind to the equilibrated resin (1 mL in batch) for 20 min at room temperature, after which the resin was washed in TPSG, pH 7.0, at 4–8 °C, followed by a 5-min centrifugation at 700g. The washing protocol included two washes with 15 mL buffer containing 2 mM imidazole, one with 15 mL buffer containing 5 mM imidazole and, finally, one wash in buffer without Triton X-100 (PSG), but with 5 mM imidazole. The resin was subsequently placed in a plastic column (Bio-Rad) equipped with a glass fiber mesh and washed with PSG/5 mM imidazole at 4–8 °C (5 mL; wash fractions 1–5). Thereafter, the enzyme was eluted with elution buffer (40 mM NaH2 PO4 , pH 7.0, 150 mM NaCl, 5% glycerol, 1 complete protease inhibitor cocktail, and 150 mM imidazole) at 4–8 °C and 0.5-mL fractions were collected. Protein concentrations were determined employing the Bio-Rad DC Protein Assay or the Pierce BCA Protein Assay, with bovine serum albumin as standard. Enzyme fractions were either stored overnight at 4 °C (for the activity measurements shown in Table 1) or at )70 °C until further use. Determination of the activity kinetics of 11b-HSD1 Fractions from the column were incubated at 37 °C for 10 min in a 200-lL reaction mixture containing 100 mM Tris–Cl, pH 7.4, 500 lM cofactor (NADPþ or
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Table 1 Purification of recombinant human and rat 11b-HSD1 Fraction
Total protein (mg)
11b-Oxo reduction Total activity (nmol/min)
11b-HSD Specific activity (nmol/min/mg)
Yield (%)
Total activity (nmol/min)
Specific activity (nmol/min/ mg)
Yield (%)
Human Microsomes Extract Eluate 1 Eluate 2 Eluate 3
89.1 65.1 0.05 0.42 0.11
96.0 61.6 0.5 5.5 1.0
1.1 0.9 10.3 13.0 8.9
100 64.1 0.5 5.7 1.0
514 295 4.5 35.1 7.7
5.8 4.5 89.7 83.7 70.3
100 57.3 0.9 6.8 1.5
Rat Microsomes Extract Eluate 1 Eluate 2 Eluate 3
49.3 23.5 0.04 0.08 0.02
589 346 5.0 7.2 1.4
12.0 14.7 126 89.9 70.0
100 58.8 0.9 1.2 0.2
858 457 9.1 19.2 3.0
17.4 19.4 228 240 151
100 53.2 1.1 2.2 0.4
Purification was made as outlined in Fig. 2. The substrates used to analyze 11b-oxo reduction and 11b-HSD were cortisone and cortisol, or 11dehydrocorticosterone and corticosterone, for the human or the rat enzyme, respectively.
an NADPH-regenerating system; i.e., 500 lM NADPH, 5 mM glucose-6-phosphate, 100 U/mL glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides, Sigma–Aldrich)), 0.05–4 lg purified protein (or 1–20 lg microsomal protein), and 0.05–80 lM 3 H-substrate (as documented in Table 2). The reactions were performed as follows: reaction mixture containing everything but the enzyme was pre-warmed for 15 min at 37°C, while 10 lL sample was added to chilled PCR tubes (Applied Biosystems). Thereafter, 190 lL pre-warmed reaction mixture was added to the enzyme to initiate the reaction. After a 10-min incubation at 37 °C, the reaction tubes were moved to an ice-cold metal rack and 25 lL of 6 M HClO4 was added to stop the reactions. After mixing on a vortex, the tubes were centrifuged for 10 min at 1300g. The supernatants thus obtained were analyzed by reverse-phase HPLC (using a C18 column) with which cortisone and cortisol could be separated by elution with 28% acetonitrile and corticosterone and 11-dehydrocorticosterone with 33% acetonitrile. Radioactivity was measured using a FLO-ONE BETA online scintillation
counter (Packard Bioscience, Meriden, CT). The percentage conversion of substrate to product was calculated on the basis of the distribution of radioactivity between the two peaks corresponding to cortisone and cortisol, or to corticosterone and 11-dehydrocorticosterone. It was assured that product formation was linear with time and protein concentration. As an indication of the initial reaction rate, the 10-min reaction did not consume more than 20% of substrate. Kinetic constants were calculated employing the GraphPad Prism software for Windows (GraphPad Software, San Diego, CA) using non-linear regression analysis of the Michaelis–Menten curves.
Results cDNA encoding rat hepatic 11b-HSD1 (r11b-HSD1) was obtained by RT-PCR amplification of the total RNA isolated from a Sprague–Dawley rat liver. Six clones were sequenced and compared to the published
Table 2 Kinetic constants of purified recombinant human and rat 11b-HSD1 Source
11-Oxo reduction
11b-HSD
KM ðlMÞ
Vmax (nmol/min/mg)
KM ðlMÞ
Vmax (nmol/min/mg)
Human Microsomes Purified
n.d 0.6 (0.2)
n.d. 3.8 (0.4)
4.1 (0.6) 1.7 (0.2)
7.7 (0.4) 69.8 (3.1)
Rat Purified
3.1 (0.9)
42.5 (4.9)
2.5 (0.2)
89.9 (6.8)
Purification procedures are the same as in Table 1. Details on the measurement of enzymatic activity, KM and Vmax are given in Materials and methods. KM is given in lM and Vmax is given in nmol product formed per min per mg protein. Values are given as means (SD), n ¼ 2 to 3. nd ¼ not determined.
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sequence [9]. All clones that were sequenced were identical to each other, but exhibited two differences from the reported sequence: at the protein level, one amino acid difference (Gln240 to Glu) and one amino acid insertion (Ile246) (see Fig. 1A). These differences were observed in a region of 11b-HSD1, which is totally conserved in seven different species and is located close to the C-terminus of the protein (Fig. 1A). Thus, the sequence for the rat enzyme reported here (GenBank AF542063) more closely resembles the corresponding sequence for other species than does the rat sequence reported earlier [9]. The methylotrophic yeast P. pastoris was selected as the host for recombinant expression, since this yeast has been previously used successfully for expression of human 11b-HSD1 (h11b-HSD1; [11]). The plasmids pMB1250 (pPIC3.5K-based) and pMB1116 (pPICZBbased) were designed to obtain expression of full-length human and rat 11b-HSD1 with an affinity-handle fused to the N-terminus (Nhis-h11b-HSD1 and Nhis-r11bHSD1, respectively). These affinity handles were chosen because previous dye-affinity purification using the cofactor binding site yielded 11b-HSD1, exhibiting only dehydrogenase activity [4]. The N-terminal extension contains a hexa-histidine sequence (the affinity handle), followed by a cleavage site recognized by the selective picornavirus protease 3C (Fig. 1B). These expression vectors were integrated into the P. pastoris genome by homologous recombination at the AOX1 locus. In this way, transcription of the 11b-HSD1 gene was placed under the control of the methanol-inducible AOX1 promoter. During development of the fermentation procedure, an almost identical P. pastoris strain was used, which produces human 11b-HSD1 without attached affinity tag [11]. Fifteen-fold higher specific activity was obtained when clones expressing h11b-HSD1 were cultured in fermentors using a complex medium rather than in shake flasks using BMGY (and BMMY for induction). We used complex medium in the fermenters to enable short incubation times. Higher 11b-HSD1 activity was obtained by induction with 0.2% (v/v) methanol than with 0.5% methanol (data not shown). In both cases, methanol was added continuously to maintain a constant concentration as monitored by the sensor (see Materials and methods). Addition of methanol to 0.2% resulted in a slower rate of induction of protein production, starting after 8 h and reaching a maximum at 16–24 h, whereas 0.5% methanol resulted in maximal activity after 2–3 h. The use of fermentors yielded considerably higher densities of cells (OD600 ¼ 60 to 90) and a higher final mass of cells (300 g wet weight per 2.5 L) than obtained from shakeflask cultures (OD600 ¼ 4 to 8, 5 g wet weight per 3 L). Under the conditions we chose, both enzymes were produced in moderate amounts, Nhis-h11b-HSD1 and
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Nhis-r11b-HSD1 constituted approximately 3% and 5%, respectively, of total microsomal protein, as judged from SDS–PAGE analysis. When assayed in cell lysates, the recombinant enzymes displayed not only dehydrogenase, but also oxidoreductase activity (data not shown). The purification strategy (Fig. 2) was based on pilot experiments, which revealed that microsomes prepared from sonicated yeast cells displayed little or no oxidoreductase activity. The levels of oxidoreductase activity in microsomal fractions obtained using three more gentle procedures for disrupting the yeast cells were compared employing the strain producing Nhis-h11bHSD1. Spheroplast generation by zymolyase treatment yielded the highest specific activity (74 pmol/min/mg protein), followed by disintegration using a BioNeb apparatus (53 pmol/min/mg protein). The lowest specific activity (28 pmol/min/mg protein) was obtained using a Bead-beater device (glass beads). For practical reasons, the BioNeb apparatus was selected for the full-scale procedure. Three different detergents were compared in connection with solubilization of the active enzyme from microsomes. The detergents, i.e., Triton X-100, octylglucoside (OG), and CHAPS all solubilized 11bHSD1 activity. The highest amount of active enzyme was obtained with 1% Triton X-100 (713 pmol dehydrogenase activity/min/mg protein), compared to values of 573 for 50 mM OG and only 141 in the case of 10 mM CHAPS. The solubilized microsomal fraction was then subjected to Immobilized Metal Ion Affinity Chromatog-
Fig. 2. Purification strategy for recombinant 11 b-HSD1 enzymes.
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raphy (IMAC) using either the Ni2þ -based NTA matrix (from Qiagen) or the Co2þ -based TALON matrix (from Clontech). Purification could be obtained with both matrixes, but the TALON matrix yielded more homogeneous 11b-HSD1 (data not shown). Initially, the enzymes were extracted with 1% Triton X-100 at pH 7.0, and eluted from the column with imidazole-containing buffer including either 1% or 0.1% Triton X-100 (pH 7.0). However, with this approach, both oxidoreductase activity and the protein concentration were low in the eluted fractions. Based on earlier reports, the pH of the extraction buffer was changed to the more optimal 7.5 [12] and elution without detergents was attempted [4]. These changes resulted in a 3–4-fold increase in the protein concentration of the eluates and readily detectable oxidoreductase activity, even without an NADPHregenerating system. However, to avoid loss of activity during the assay, an NADPH-regenerating system was routinely included in the assay mixture. The oxidoreductase activity was more sensitive to prolonged storage at 4 °C and to repeated freezing and thawing, than was the dehydrogenase activity, in agreement with earlier reports [12]. Densitometric analysis following SDS–PAGE indicated that both enzymes were 90–95% pure (Fig. 3). Contaminating bands were present to a small extent, although none more prominent than others, suggesting that no proteins were selectively co-purified together with 11b-HSD1. Both enzymes exhibited an apparent molecular weight of approximately 31 kDa. This is lower than the apparent molecular weight of 11b-HSD1 expressed in mammalian cells (34 kDa; [5]), but in agreement with previous work by Blum et al. [13] who demonstrated that h11b-HSD1
produced in P. pastoris behaves electrophoretically in the same way as the deglycosylated hepatic enzyme. The specific activities of the two enzymes were monitored at each step of the purification procedure (Table 1). The oxidoreductase activity of both human and rat 11bHSD1 was increased by a factor of approximately 10 upon purification by IMAC. The dehydrogenase activity increased somewhat more, i.e., 15- and 12-fold, respectively, for the human and rat enzymes. These values are low in relationship to the purity of 90–95%, indicated by SDS–PAGE (Fig. 3), and suggest that enzyme activity is lost in the final purification step. However, this loss appears to affect the reactions in both directions to a similar extent and is, thus, not a unidirectional loss of activity, as observed with previous purification protocols [4]. The kinetic constants of the purified enzymes were subsequently determined (Table 2). The purified NHish11b-HSD1 displayed a slightly lower KM for cortisol than did the membrane-bound enzyme. The specific oxidoreductase activity (Vmax ) of the purified human enzyme was considerably lower (15-fold) than the dehydrogenase activity, in agreement with a previous report on h11b-HSD1 expressed in yeast microsomes [11]. The human enzyme exhibited a slightly higher affinity for cortisone than for cortisol, reflecting its primary reaction in vivo. NHis-r11b-HSD1 had a lower affinity for its physiological substrate (11-dehydrocorticosterone) than did the human recombinant enzyme (KM values of 3.1 and 0.6 lM, respectively). The specific oxidoreductase activity (Vmax ) was found to be 10-fold higher for the rat than for the human enzyme (Table 2).
Fig. 3. Purification of human and rat 11b-HSD1 by cobalt affinity chromatography. (A) Purification of human Nhis-11b-HSD1 by cobalt ion affinity chromatography from P. pastoris microsomes, as outlined in Fig. 2. Lanes 1 ¼ microsomes (110 lg), 2 ¼ Triton X-100 extract (20 lg), 3 ¼ Novex Mark12 Mw marker, 4 ¼ flowthrough (0.07% v/v), 5–6 ¼ first + last wash (0.1% v/v), and 7–9 ¼ elutions (2% v/v). The samples were run on a 10% acrylamide (Bis-Tris-MOPS buffered) NuPAGE (NOVEX, Invitrogen) and subsequently stained with coomassie blue. (B) Purification of rat Nhis11b-HSD1 by cobalt ion affinity chromatography from P. pastoris microsomes, as outlined in Fig. 2. Lanes 1 ¼ microsomes (90 lg), 2–3 ¼ Triton X100 extract (20 lg), 4 ¼ Novex Mark12 Mw marker, 5 ¼ flowthrough (0.07% v/v), 6 ¼ last wash (0.1% v/v), and 7–8 ¼ elution (2% v/v). Samples were run on a 4–12% (Bis-Tris-MES buffered) NuPAGE (NOVEX) and subsequently stained with Coomassie blue.
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Discussion The principle of local inactivation and reactivation of steroids and other hormones is now well established (reviewed in [14]). The general theme of separate enzymes catalyzing the forward and reverse reactions was first established for sex hormones, which are inactivated and reactivated by separate 17b-hydroxysteroid dehydrogenases [15]. The principle of local tissue metabolism also applies to glucocorticoid hormones, where inactivation of cortisol (and corticosterone) by 11b-HSD type 2 is undisputed [1]. Originally, 11b-HSD1 was believed to be a dehydrogenase, since Lakshmi and Monder [4] could detect only dehydrogenase activity when they first purified the enzyme from rat liver in 1988. It was later demonstrated that 11b-HSD1 functions primarily as an oxidoreductase in intact cells, e.g., in the liver [16], adipocytes [17], and neurons [18]. The fact that the dehydrogenase activity of 11bHSD1 is much more stable in vitro is intriguing. Previous data suggest that factors present in the intracellular environment are important for the oxidoreductase activity. Agarwal et al. [9] found that regeneration of NADPH employing glucose-6-phosphate dehydrogenase and glucose-6-phosphate enhances the oxidoreductase activity, although this activity was still found to be less stable than the dehydrogenase activity. Detailed studies on kinetic mechanisms and posttranscriptional regulation require a highly purified enzyme. Most previous attempts to purify native 11bHSD1, e.g., from rat [4], rabbit [19], guinea pig [20], and human [21] have confirmed the instability of the oxidoreductase activity in vitro. In one study, 11b-HSD1 purified from mouse liver retained oxidoreductase activity, but the KM of this reaction was reported to be 220 lM [22]. This is surprisingly high, not only in comparison to the KM values of 0.6 and 3.1 lM obtained in the present study, for the enzymes from human and rat, respectively, but also in comparison with KM values obtained using more crude extracts [5,11,23]. In a recent study, a truncated form of human 11b-HSD1 expressed in E. coli was reported to display both dehydrogenase and oxidoreductase activities [24]. However, no details on the latter activity were given, except that the KM value was 9.5 lM. The strategy behind the purification procedure developed here was to avoid affinity chromatography using either cofactor or substrate as ligand, since previous reports indicate that no oxidoreductase activity is recovered using such ligands [4,17,18,20]. To this end, recombinant enzymes were designed to contain an affinity handle, which would enable affinity purification without disturbing the enzymatic activity. Thus, the hexa-histidine affinity handle was attached to the Nterminus of 11b-HSD1, which in vivo is situated at the
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surface of the endoplasmic membrane, opposite to that of the catalytic domain from which it is separated by a transmembrane segment containing approximately 20 amino acids [3,25]. The data presented here indicate that this affinity handle did not affect the catalytic activities of the enzymes (Table 1). Furthermore, our data strongly suggest that gentle disruption of the yeast cells is important for retention of the oxidoreductase activity. This finding might explain some of the earlier failures in obtaining active enzyme upon purification from yeast or bacteria disrupted using harsher procedures [13,26] and is in agreement with the conclusions reached by Walker et al. [24]. In addition, more oxidoreductase activity was recovered when detergent was omitted from the elution buffer employed for the affinity purification. This observation was unexpected, considering the fact that the protein contains a hydrophobic membrane anchor. Both this finding and the apparent sensitivity to sonication might be explained by dissociation of aggregates of significance for the oxidoreductase activity. For example, rat 11b-HSD1 has been shown to form aggregates of 170 or 340 kDa, with and without detergent, respectively [4]. The catalytic properties of purified human and rat 11b-HSD1 determined here were similar to those reported earlier. For example, our recombinant Nhisr11b-HSD1 displays a KM of 2.5 lM for 11-dehydrocorticosterone, which is comparable to the KM value of 1.8 lM for native rat 11b-HSD1 [4]. Furthermore, our enzyme has a KM for corticosterone of 3.1 lM, which is comparable to the corresponding value of 2.8 lM reported for recombinant rat enzyme assayed in cell extracts [5]. However, direct comparisons of values cannot be made, since different investigators have used different pH values in their assay medium. Here, a physiological pH of 7.4 was employed. In conclusion, expression of an appropriately tagged protein in yeast, followed by a simple single-step affinity purification procedure, has been successfully developed here to obtain full-length 11b-HSD1 with retention of oxidoreductase activity. This procedure is equally effective with 11b-HSD1 from both human and rat. This successful purification will facilitate future detailed biochemical and biophysical characterization of this important enzyme.
Acknowledgments ke We are grateful to Andrea Varadi, Sven-A Franzen, and Marianne Israelsson for DNA sequencing, to Margareta Forsgren for cloning of the human 11bHSD1 and for technical advice, and to Camilla Sivertsson for help with the fermentor cultures. Drs. Udo Oppermann and Joakim Nilsson are acknowledged for fruitful discussions and helpful comments on the
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manuscript. We also want to thank Professor Joe de Pierre for help with the manuscript. C.S.I. Nobel acknowledges funding for a postdoctoral position by Pharmacia & Upjohn AB and grants from the Department of Biochemistry and Biophysics at Stockholm University, Carl TryggerÕs Stiftelse, and Helge Ax:son JohnsonÕs Stiftelse.
References [1] P.M. Stewart, Z.S. Krozowski, 11ß-Hydroxysteroid dehydrogenase, Vitam. Horm. 57 (1999) 249–324. [2] H. Masuzaki, J. Paterson, H. Shinyama, N.M. Morton, J.J. Mullins, J.R. Seckl, J.S. Flier, A transgenic model of visceral obesity and the metabolic syndrome, Science 294 (2001) 2166– 2170. [3] U.C. Oppermann, B. Persson, H. Jornvall, Function, gene organization and protein structures of 11b-hydroxysteroid dehydrogenase isoforms, Eur. J. Biochem. 249 (1997) 355–360. [4] V. Lakshmi, C. Monder, Purification and characterization of the corticosteroid 11b-dehydrogenase component of the rat liver 11bhydroxysteroid dehydrogenase complex, Endocrinology 123 (1988) 2390–2398. [5] A.K. Agarwal, M.T. Tusie-Luna, C. Monder, P.C. White, Expression of 11b-hydroxysteroid dehydrogenase using recombinant vaccinia virus, Mol. Endocrinol. 4 (1990) 1827–1832. [6] G.M. Tannin, A.K. Agarwal, C. Monder, M.I. New, P.C. White, The human gene for 11b-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization, J. Biol. Chem. 266 (1991) 16653–16658. [7] J. Nilsson, S. Stahl, J. Lundeberg, M. Uhlen, P.A. Nygren, Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins, Protein Expr. Purif. 11 (1997) 1– 16. [8] H. Miyasaka, The positive relationship between codon usage bias and translation initiation AUG context in Saccharomyces cerevisiae, Yeast 15 (1999) 633–637. [9] A.K. Agarwal, C. Monder, B. Eckstein, P.C. White, Cloning and expression of rat cDNA encoding corticosteroid 11b-dehydrogenase, J. Biol. Chem. 264 (1989) 18939–18943. [10] K.N. Faber, Y. Elgersma, J.A. Heyman, A. Koller, G.H. Luers, W.M. Nuttley, S.R. Terlecky, T.J. Wenzel, S. Subramani, Use of Pichia pastoris as a model eukaryotic system. Peroxisome biogenesis, Methods Mol. Biol. 103 (1998) 121–147. [11] M. Hult, H. Jornvall, U.C. Oppermann, Selective inhibition of human type 1 11b-hydroxysteroid dehydrogenase by synthetic steroids and xenobiotics, FEBS Lett. 441 (1998) 25–28. [12] V. Lakshmi, C. Monder, Extraction of 11b-hydroxysteroid dehydrogenase from rat liver microsomes by detergents, J. Steroid Biochem. 22 (1985) 331–340.
[13] A. Blum, H.J. Martin, E. Maser, Human 11b-hydroxysteroid dehydrogenase type 1 is enzymatically active in its nonglycosylated form, Biochem. Biophys. Res. Commun. 276 (2000) 428– 434. [14] S. Nobel, L. Abrahmsen, U. Oppermann, Metabolic conversion as a pre-receptor control mechanism for lipophilic hormones, Eur. J. Biochem. 268 (2001) 4113–4125. [15] F. Labrie, Intracrinology, Mol. Cell. Endocrinol. 78 (1991) C113– C118. [16] P.M. Jamieson, K.E. Chapman, C.R. Edwards, J.R. Seckl, 11bHydroxysteroid dehydrogenase is an exclusive 11b-reductase in primary cultures of rat hepatocytes: effect of physicochemical and hormonal manipulations, Endocrinology 136 (1995) 4754– 4761. [17] I.J. Bujalska, S. Kumar, P.M. Stewart, Does central obesity reflect ‘‘CushingÕs disease of the omentum’’?, Lancet 349 (1997) 1210– 1213. [18] V. Rajan, C.R. Edwards, J.R. Seckl, 11b-Hydroxysteroid dehydrogenase in cultured hippocampal cells reactivates inert 11dehydrocorticosterone, potentiating neurotoxicity, J. Neurosci. 16 (1996) 65–70. [19] J. Ozols, Lumenal orientation and post-translational modifications of the liver microsomal 11b-hydroxysteroid dehydrogenase, J. Biol. Chem. 270 (1995) 2305–2312. [20] U.C. Oppermann, E. Mobus, G. Nagel, E. Maser, Heterogeneity of 11b-hydroxysteroid dehydrogenase type1/microsomal carbonyl reductase (11b-HSD/CR) in guinea pig tissues. Purification of the liver form suggests modification in the cosubstrate binding site, Toxicology 144 (2000) 63–69. [21] M. Hult, C.S. Nobel, L. Abrahmsen, D.A. Nicoll-Griffith, H. Jornvall, U.C. Oppermann, Novel enzymological profiles of human 11b-hydroxysteroid dehydrogenase type 1, Chem. Biol. Interact. 130–132 (2001) 805–814. [22] E. Maser, G. Bannenberg, The purification of 11b-hydroxysteroid dehydrogenase from mouse liver microsomes, J. Steroid Biochem. Mol. Biol. 48 (1994) 257–263. [23] S. Diederich, C. Grossmann, B. Hanke, M. Quinkler, M. Herrmann, V. B€ahr, W. Oelkers, In the search for specific inhibitors of human 11b-hydroxysteroid-dehydrogenases (11bHSDs): chenodeoxycholic acid selectively inhibits 11b-HSDI, Eur. J. Endocrinol. 142 (2000) 200–207. [24] E.A. Walker, A.M. Clark, M. Hewison, J.P. Ride, P.M. Stewart, Functional expression, characterization, and purification of the catalytic domain of human 11b-hydroxysteroid dehydrogenase type 1, J. Biol. Chem. 276 (2001) 21343–21350. [25] A. Odermatt, P. Arnold, A. Stauffer, B.M. Frey, F.J. Frey, The Nterminal anchor sequences of 11b-hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane, J. Biol. Chem. 274 (1999) 28762–28770. [26] A. Blum, H.J. Martin, E. Maser, Human 11b-hydroxysteroid dehydrogenase1/carbonyl reductase: recombinant expression in the yeast Pichia pastoris and Escherichia coli, Toxicology 144 (2000) 113–120.
Purification of full-length recombinant human and rat type 1 11b-hydroxysteroid dehydrogenases with retained oxidoreductase activities C. Stefan I. Nobel,a,b Finn Dun as,a,1 and Lars. B. Abrahmsena,* a b
Biovitrum AB, Division of Pharmaceuticals, Department of Assay Development and Screening, S-112 87 Stockholm, Sweden Unit for Biochemical Toxicology, Department of Biochemistry and Biophysics, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden Received 6 February 2002, and in revised form 8 July 2002
Abstract 11b-Hydroxysteroid dehydrogenase type 1 (11b-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum. This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase. Previous attempts to purify the native enzyme have yielded a protein without reductase activity. To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11b-HSD1 with retention of their natural oxidoreductase activities. This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography. Both enzymes were 90–95% pure and exhibited dehydrogenase and reductase activities with KM values in agreement with those reported in the literature. Ó 2002 Elsevier Science (USA). All rights reserved.
11b-Hydroxysteroid dehydrogenase type 1 (11bHSD1) plays a key role in regulating local tissue glucocorticoid concentrations by converting cortisone into the active glucocorticoid cortisol in humans and 11-dehydrocorticosterone into corticosterone in rodents (reviewed in [1]). Gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels and for the up-regulation of key hepatic gluconeogenic enzymes. This enzyme is expressed primarily in the liver, adipose tissue, brain, gonads, and blood vessels [1]. The phenotype of transgenic mice in which 11b-HSD1 is over-expressed in adipose tissue indicates that this enzyme plays a role in connection with syndrome X and diabetes [2]. Native 11b-HSD1 is a member of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins *
Corresponding author. E-mail address: [email protected] (L.B. Abrahmsen). 1 Present address: Affibody AB, P.O. Box 20137, S-16102 Bromma, Sweden.
(reviewed in [3]). This 34 kDa, glycosylated, NADP(H)dependent enzyme is attached to the endoplasmic reticular membrane and faces the lumenal compartment. The enzyme is bidirectional in vitro, but is believed to predominantly function as an oxidoreductase in vivo [1]. Surprisingly, the oxidoreductase activity of 11b-HSD1 is more sensitive to inactivation than is the dehydrogenase activity, both in cell extracts and following purification of the enzyme [4,5]. This unexpected property prompted us to investigate the catalytic mechanism of 11b-HSD1 in greater detail. To enable such studies, we decided to purify the recombinant enzyme. Recombinant fulllength 11b-HSD1 has not been purified previously and when purified from natural sources only dehydrogenase activity has been retained. Materials and methods Materials Chemicals and reagents were purchased from Sigma– Aldrich (Stockholm, Sweden) or Merck Eurolab
1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 5 9 2 8 ( 0 2 ) 0 0 5 4 7 - 8
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(Stockholm, Sweden), if not otherwise stated. [1,23 H(N)]-Hydrocortisone, 52 Ci/mmol, was obtained from NEN Life Sciences (Zaventem, Belgium), while [1,2,6 7-(3 H)]-corticosterone, 77 Ci/mmol, and the customsynthesized [1,2(n)-3 H]-cortisone, 46 Ci/mmol, and 11dehydro-[3 H]-corticosterone, 61 Ci/mmol, were supplied by Amersham Pharmacia Biotech (Amersham, UK). Oligonucleotides were custom-made by Life Technologies (Paisley, Scotland) and DNA plasmid purification kits were purchased from Qiagen (Hilden, Germany). Cloning of 11b-HSD1 and generation of yeast clones expressing this enzyme The human 11b-HSD1 gene was cloned from HepG2 cells by RT-PCR and found to encode a protein with a primary sequence identical to the published one [6]. An N-terminal 6His tag containing a cleavage site for picornavirus protease 3C [7] was fused to the hydrophobic anchoring domain of the enzyme by employing two consecutive PCRs (using the Expand High Fidelity PCR system from Roche Diagnostics, Bromma, Sweden, according to supplierÕs protocol and with an annealing temperature of 55 °C). The primers used for the first amplification were forward 1: 50 -TTA GAA GCT TTA TTT CAA GGG CCA ATG GCT TTT ATG AAA AAA TAT CTC CTC CCC ATT CTG GG-30 and reverse 1: 50 -GCG CGG CCG CCT ACT TGT TTA TGA ATC TGT CCA TAT TAT AGC TCG-30 ); while the primers for the second reaction were forward 2: 50 -CGG AAT TCG GTA CC TAC AAA ATG TCT CAC CAT CAC CAT CAC CAT TTA GAA GCT TTA TTT CAA GGG CC-30 and reverse 1 (see above). The final PCR product coded for human 11b-HSD1, preceded by the amino acid sequence MSHHHHHHLE ALFQGP (Fig. 1) and contains sites for EcoRI and KpnI as well as a yeast optimal translation start sequence TACAAAATG at the 50 -end [8]. The reverse primer introduced a NotI site after the stop codon. This construct was inserted between EcoRI and NotI in the vector pPIC3.5K (Invitrogen, Groningen, The Netherlands), to obtain pMB1250. This recombinant plasmid DNA was linearized with PmeI and transformed into the Pichia pastoris strain GS115 according to supplierÕs protocol (Invitrogen). Rat 11bHSD-1 was obtained by RT-PCR employing total RNA from Sprague–Dawley rat liver (kit from Perkin–Elmer, Applied Biosystems, Stockholm, Sweden; with an annealing temperature of 55 °C). The primers (forward 3: 50 -CG GAA TTC GGT ACC GCC GCC ACC ATG AAA AAA TAC CTC CTC CCC G-30 and reverse 2: 50 -GC TCT AGA GCG GCC GC GTT GCT TAC AAA TAG GTC CCT G-30 ) added sites for EcoRI, KpnI, and a Kozak sequence at the 50 -end, and removed the stop codon and added NotI and XbaI sites at the 30 end. The PCR product was inserted into pPICZaA (In-
Fig. 1. Updated rat 11b-HSD1 sequence and sequence of constructs used for purification. (A) Amino acid sequence alignment of published 11b-HSD1 orthologs. The conserved motif (KEECALEIIKG) near the C-terminal is not contained in the published rat 11b-HSD1 sequence (deviations towards other 11b-HSDs highlighted), but is found in the one reported here. (Squirrel monkey is abbreviated Sq. monkey). (B) The N-terminal modification of both rat and human 11b-HSD1 including the 6His sequence and the Picornavirus 3C protease site (underlined). Sequence in bold represents the amino-terminal portion of human 11b-HSD1.
vitrogen) employing EcoRI and NotI to yield pMB1020. This plasmid adds the yeast secretory a-factor at the Nterminus plus a myc epitope and a 6His tag at the C-terminus of rat 11b-HSD1. Six of the clones were sequenced (cycle sequencing using BigDye Terminator Kit from Applied Biosystems) and compared to the published rat 11b-HSD1 DNA sequence [9] (see text for details). The plasmid pMB1020 was the template in a subsequent PCR (Expand High Fidelity PCR system from Roche Diagnostics) performed at an annealing temperature of 55 °C with a new set of primers (forward 4: 50 CG GAA TTC TAC AAA ATG TCT CAC CAT CAC CAT CAC CAT TTA GAA GCT TTA TTT CAA GGG CCA ATG AAA AAA TAC C-30 , reverse 3: 50 GC GCG GCC GCT CAG TTG CTT ACA AAT AGG TCC CTG-30 ). This introduced the same modification of the protein as in the case of human 11bHSD-1 (see above). The PCR product was inserted between EcoRI and NotI in pPICZB (Invitrogen) to yield pMB1116. This plasmid was linearized with PmeI and transformed into the P. pastoris strain XL33 (Invitrogen) using Zeocin as the selecting agent for integration into the genome according to supplierÕs protocol (Invitrogen). Expression in yeast The recombinant P. pastoris strains GS115 and XL33 containing genes encoding N-terminal 6His (N-His)-
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tagged human 11b-HSD1 and rat 11b-HSD1, respectively, exhibited Mutþ (methanol using plus) phenotypes. One colony from a fresh agar plate (grown in minimal dextrose medium (MD) agar plate in the case of the former strain and in yeast extract peptone containing Zeocin in the case of the latter strain), was transferred into 50 mL BMGY and incubated at 30 °C for 15 h in a 300-mL Tunair flask with shaking (Shelton Scientific, Shelton, CT). BMGY contains 1% yeast extract (Merck), 2% peptone (Difco, Detroit, MI), 100 mM potassium phosphate, pH 6.0, 1.34% yeast nitrogen base (YNB), 1% glycerol, and 4 105 % biotin. The culture was then transferred into 200 mL fresh BMGY in a 2.5-L Tunair flask and incubated for an additional 6 h with shaking. A fermentor (3-L working volume, Belach Bioteknik AB, Sweden) containing the components of BMGY (but with less phosphate buffer, i.e., 42 mM, and more glycerol, (4%)) and 0.5 mL/L of an antifoaming agent (Adekanol) was in situ sterilized. Filter-sterilized YNB and biotin were subsequently added. A probe connected to a methanol sensor (Raven Biotech, Canada) was then aseptically mounted onto the fermentor. The fermentor was aerated with 1 vvm air and the dissolved oxygen tension (DOT) was maintained at 40% atmospheric pressure by mixing. In some cases, a 1:1 air/oxygen mixture was used towards the end of the growth period to achieve a sufficiently high DOT. The temperature was set at 30 °C and the pH was maintained at 6 by automatic addition of 25% ammonia when required. Addition of 50% glycerol (20–30 g/L h, containing 1.2% PTM1 trace element solution; Invitrogen) was initiated when the original glycerol had been depleted (as indicated by a peak signal in the DOT). After a few hours of such addition, glycerol was replaced by methanol containing 1.2% PTM1, which was pumped manually into the fermentor until a concentration of 0.2% (v/v) had been reached. The methanol sensor was used to maintain the methanol concentration at same level throughout cultivation. The cultures were harvested approximately 16–24 h after the initial addition of methanol, at which point the OD600 was 60–90. One hundred grams cell aliquots were dissolved in 100 mL buffer (20 mM NaH2 PO4 , pH 7, with 15% glycerol) and frozen at )20 °C until use for the preparation of microsomes. Preparation of microsomes Suspensions containing 100 g cells were diluted to 0.3 g/mL with buffer, giving a final concentration of 20 mM NaH2 PO4 , pH 7, and 9% glycerol as well as 1:5 complete protease inhibitor cocktail (Roche Diagnostics). Microsomes were produced by processing this cell suspension in a BioNeb Cell disintegrator (Glas-Col, Terre Haute, IN), under nitrogen gas at a pressure of 11
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bars (60–90 min per 167 mL (50 g) cells in continuous recycling mode). In pilot experiments, yeast cells were disrupted employing the zymolyase procedure to obtain spheroplasts [10]. The cell homogenate was centrifuged at 9000g for 15 min and the pellet was discarded. The resulting supernatant was centrifuged at 140,000g for 60 min and the pellet (microsomes) resuspended in buffer (40 mM NaH2 PO4 , pH 7.5, 500 mM NaCl, 10% glycerol, and 1 complete protease cocktail) using a homogenizer with a Teflon pestle and then frozen in aliquots at )70 °C. Microsomal protein content was determined using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA) or the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL). Purification of 11b-HSD1 Frozen microsomes were thawed on ice and diluted to 10 mg/mL in TPSG extraction buffer (containing 40 mM NaH2 PO4 , pH 7.5, 500 mM NaCl, 5% glycerol, 1% Triton X-100, and 1 complete protease inhibitor cocktail). Microsomal protein was solubilized by gently mixing this suspension for 30 min at 4–8 °C. The mixture was then centrifuged at 105,000g for 60 min and the pellet was discarded. Subsequently, chromatography was performed using a TALON cobalt ion-based resin (BD Biosciences Clontech, Palo Alto, CA), according to manufacturerÕs instructions. Briefly, the solubilized microsomal fraction (10 mL) was allowed to bind to the equilibrated resin (1 mL in batch) for 20 min at room temperature, after which the resin was washed in TPSG, pH 7.0, at 4–8 °C, followed by a 5-min centrifugation at 700g. The washing protocol included two washes with 15 mL buffer containing 2 mM imidazole, one with 15 mL buffer containing 5 mM imidazole and, finally, one wash in buffer without Triton X-100 (PSG), but with 5 mM imidazole. The resin was subsequently placed in a plastic column (Bio-Rad) equipped with a glass fiber mesh and washed with PSG/5 mM imidazole at 4–8 °C (5 mL; wash fractions 1–5). Thereafter, the enzyme was eluted with elution buffer (40 mM NaH2 PO4 , pH 7.0, 150 mM NaCl, 5% glycerol, 1 complete protease inhibitor cocktail, and 150 mM imidazole) at 4–8 °C and 0.5-mL fractions were collected. Protein concentrations were determined employing the Bio-Rad DC Protein Assay or the Pierce BCA Protein Assay, with bovine serum albumin as standard. Enzyme fractions were either stored overnight at 4 °C (for the activity measurements shown in Table 1) or at )70 °C until further use. Determination of the activity kinetics of 11b-HSD1 Fractions from the column were incubated at 37 °C for 10 min in a 200-lL reaction mixture containing 100 mM Tris–Cl, pH 7.4, 500 lM cofactor (NADPþ or
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Table 1 Purification of recombinant human and rat 11b-HSD1 Fraction
Total protein (mg)
11b-Oxo reduction Total activity (nmol/min)
11b-HSD Specific activity (nmol/min/mg)
Yield (%)
Total activity (nmol/min)
Specific activity (nmol/min/ mg)
Yield (%)
Human Microsomes Extract Eluate 1 Eluate 2 Eluate 3
89.1 65.1 0.05 0.42 0.11
96.0 61.6 0.5 5.5 1.0
1.1 0.9 10.3 13.0 8.9
100 64.1 0.5 5.7 1.0
514 295 4.5 35.1 7.7
5.8 4.5 89.7 83.7 70.3
100 57.3 0.9 6.8 1.5
Rat Microsomes Extract Eluate 1 Eluate 2 Eluate 3
49.3 23.5 0.04 0.08 0.02
589 346 5.0 7.2 1.4
12.0 14.7 126 89.9 70.0
100 58.8 0.9 1.2 0.2
858 457 9.1 19.2 3.0
17.4 19.4 228 240 151
100 53.2 1.1 2.2 0.4
Purification was made as outlined in Fig. 2. The substrates used to analyze 11b-oxo reduction and 11b-HSD were cortisone and cortisol, or 11dehydrocorticosterone and corticosterone, for the human or the rat enzyme, respectively.
an NADPH-regenerating system; i.e., 500 lM NADPH, 5 mM glucose-6-phosphate, 100 U/mL glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides, Sigma–Aldrich)), 0.05–4 lg purified protein (or 1–20 lg microsomal protein), and 0.05–80 lM 3 H-substrate (as documented in Table 2). The reactions were performed as follows: reaction mixture containing everything but the enzyme was pre-warmed for 15 min at 37°C, while 10 lL sample was added to chilled PCR tubes (Applied Biosystems). Thereafter, 190 lL pre-warmed reaction mixture was added to the enzyme to initiate the reaction. After a 10-min incubation at 37 °C, the reaction tubes were moved to an ice-cold metal rack and 25 lL of 6 M HClO4 was added to stop the reactions. After mixing on a vortex, the tubes were centrifuged for 10 min at 1300g. The supernatants thus obtained were analyzed by reverse-phase HPLC (using a C18 column) with which cortisone and cortisol could be separated by elution with 28% acetonitrile and corticosterone and 11-dehydrocorticosterone with 33% acetonitrile. Radioactivity was measured using a FLO-ONE BETA online scintillation
counter (Packard Bioscience, Meriden, CT). The percentage conversion of substrate to product was calculated on the basis of the distribution of radioactivity between the two peaks corresponding to cortisone and cortisol, or to corticosterone and 11-dehydrocorticosterone. It was assured that product formation was linear with time and protein concentration. As an indication of the initial reaction rate, the 10-min reaction did not consume more than 20% of substrate. Kinetic constants were calculated employing the GraphPad Prism software for Windows (GraphPad Software, San Diego, CA) using non-linear regression analysis of the Michaelis–Menten curves.
Results cDNA encoding rat hepatic 11b-HSD1 (r11b-HSD1) was obtained by RT-PCR amplification of the total RNA isolated from a Sprague–Dawley rat liver. Six clones were sequenced and compared to the published
Table 2 Kinetic constants of purified recombinant human and rat 11b-HSD1 Source
11-Oxo reduction
11b-HSD
KM ðlMÞ
Vmax (nmol/min/mg)
KM ðlMÞ
Vmax (nmol/min/mg)
Human Microsomes Purified
n.d 0.6 (0.2)
n.d. 3.8 (0.4)
4.1 (0.6) 1.7 (0.2)
7.7 (0.4) 69.8 (3.1)
Rat Purified
3.1 (0.9)
42.5 (4.9)
2.5 (0.2)
89.9 (6.8)
Purification procedures are the same as in Table 1. Details on the measurement of enzymatic activity, KM and Vmax are given in Materials and methods. KM is given in lM and Vmax is given in nmol product formed per min per mg protein. Values are given as means (SD), n ¼ 2 to 3. nd ¼ not determined.
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sequence [9]. All clones that were sequenced were identical to each other, but exhibited two differences from the reported sequence: at the protein level, one amino acid difference (Gln240 to Glu) and one amino acid insertion (Ile246) (see Fig. 1A). These differences were observed in a region of 11b-HSD1, which is totally conserved in seven different species and is located close to the C-terminus of the protein (Fig. 1A). Thus, the sequence for the rat enzyme reported here (GenBank AF542063) more closely resembles the corresponding sequence for other species than does the rat sequence reported earlier [9]. The methylotrophic yeast P. pastoris was selected as the host for recombinant expression, since this yeast has been previously used successfully for expression of human 11b-HSD1 (h11b-HSD1; [11]). The plasmids pMB1250 (pPIC3.5K-based) and pMB1116 (pPICZBbased) were designed to obtain expression of full-length human and rat 11b-HSD1 with an affinity-handle fused to the N-terminus (Nhis-h11b-HSD1 and Nhis-r11bHSD1, respectively). These affinity handles were chosen because previous dye-affinity purification using the cofactor binding site yielded 11b-HSD1, exhibiting only dehydrogenase activity [4]. The N-terminal extension contains a hexa-histidine sequence (the affinity handle), followed by a cleavage site recognized by the selective picornavirus protease 3C (Fig. 1B). These expression vectors were integrated into the P. pastoris genome by homologous recombination at the AOX1 locus. In this way, transcription of the 11b-HSD1 gene was placed under the control of the methanol-inducible AOX1 promoter. During development of the fermentation procedure, an almost identical P. pastoris strain was used, which produces human 11b-HSD1 without attached affinity tag [11]. Fifteen-fold higher specific activity was obtained when clones expressing h11b-HSD1 were cultured in fermentors using a complex medium rather than in shake flasks using BMGY (and BMMY for induction). We used complex medium in the fermenters to enable short incubation times. Higher 11b-HSD1 activity was obtained by induction with 0.2% (v/v) methanol than with 0.5% methanol (data not shown). In both cases, methanol was added continuously to maintain a constant concentration as monitored by the sensor (see Materials and methods). Addition of methanol to 0.2% resulted in a slower rate of induction of protein production, starting after 8 h and reaching a maximum at 16–24 h, whereas 0.5% methanol resulted in maximal activity after 2–3 h. The use of fermentors yielded considerably higher densities of cells (OD600 ¼ 60 to 90) and a higher final mass of cells (300 g wet weight per 2.5 L) than obtained from shakeflask cultures (OD600 ¼ 4 to 8, 5 g wet weight per 3 L). Under the conditions we chose, both enzymes were produced in moderate amounts, Nhis-h11b-HSD1 and
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Nhis-r11b-HSD1 constituted approximately 3% and 5%, respectively, of total microsomal protein, as judged from SDS–PAGE analysis. When assayed in cell lysates, the recombinant enzymes displayed not only dehydrogenase, but also oxidoreductase activity (data not shown). The purification strategy (Fig. 2) was based on pilot experiments, which revealed that microsomes prepared from sonicated yeast cells displayed little or no oxidoreductase activity. The levels of oxidoreductase activity in microsomal fractions obtained using three more gentle procedures for disrupting the yeast cells were compared employing the strain producing Nhis-h11bHSD1. Spheroplast generation by zymolyase treatment yielded the highest specific activity (74 pmol/min/mg protein), followed by disintegration using a BioNeb apparatus (53 pmol/min/mg protein). The lowest specific activity (28 pmol/min/mg protein) was obtained using a Bead-beater device (glass beads). For practical reasons, the BioNeb apparatus was selected for the full-scale procedure. Three different detergents were compared in connection with solubilization of the active enzyme from microsomes. The detergents, i.e., Triton X-100, octylglucoside (OG), and CHAPS all solubilized 11bHSD1 activity. The highest amount of active enzyme was obtained with 1% Triton X-100 (713 pmol dehydrogenase activity/min/mg protein), compared to values of 573 for 50 mM OG and only 141 in the case of 10 mM CHAPS. The solubilized microsomal fraction was then subjected to Immobilized Metal Ion Affinity Chromatog-
Fig. 2. Purification strategy for recombinant 11 b-HSD1 enzymes.
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raphy (IMAC) using either the Ni2þ -based NTA matrix (from Qiagen) or the Co2þ -based TALON matrix (from Clontech). Purification could be obtained with both matrixes, but the TALON matrix yielded more homogeneous 11b-HSD1 (data not shown). Initially, the enzymes were extracted with 1% Triton X-100 at pH 7.0, and eluted from the column with imidazole-containing buffer including either 1% or 0.1% Triton X-100 (pH 7.0). However, with this approach, both oxidoreductase activity and the protein concentration were low in the eluted fractions. Based on earlier reports, the pH of the extraction buffer was changed to the more optimal 7.5 [12] and elution without detergents was attempted [4]. These changes resulted in a 3–4-fold increase in the protein concentration of the eluates and readily detectable oxidoreductase activity, even without an NADPHregenerating system. However, to avoid loss of activity during the assay, an NADPH-regenerating system was routinely included in the assay mixture. The oxidoreductase activity was more sensitive to prolonged storage at 4 °C and to repeated freezing and thawing, than was the dehydrogenase activity, in agreement with earlier reports [12]. Densitometric analysis following SDS–PAGE indicated that both enzymes were 90–95% pure (Fig. 3). Contaminating bands were present to a small extent, although none more prominent than others, suggesting that no proteins were selectively co-purified together with 11b-HSD1. Both enzymes exhibited an apparent molecular weight of approximately 31 kDa. This is lower than the apparent molecular weight of 11b-HSD1 expressed in mammalian cells (34 kDa; [5]), but in agreement with previous work by Blum et al. [13] who demonstrated that h11b-HSD1
produced in P. pastoris behaves electrophoretically in the same way as the deglycosylated hepatic enzyme. The specific activities of the two enzymes were monitored at each step of the purification procedure (Table 1). The oxidoreductase activity of both human and rat 11bHSD1 was increased by a factor of approximately 10 upon purification by IMAC. The dehydrogenase activity increased somewhat more, i.e., 15- and 12-fold, respectively, for the human and rat enzymes. These values are low in relationship to the purity of 90–95%, indicated by SDS–PAGE (Fig. 3), and suggest that enzyme activity is lost in the final purification step. However, this loss appears to affect the reactions in both directions to a similar extent and is, thus, not a unidirectional loss of activity, as observed with previous purification protocols [4]. The kinetic constants of the purified enzymes were subsequently determined (Table 2). The purified NHish11b-HSD1 displayed a slightly lower KM for cortisol than did the membrane-bound enzyme. The specific oxidoreductase activity (Vmax ) of the purified human enzyme was considerably lower (15-fold) than the dehydrogenase activity, in agreement with a previous report on h11b-HSD1 expressed in yeast microsomes [11]. The human enzyme exhibited a slightly higher affinity for cortisone than for cortisol, reflecting its primary reaction in vivo. NHis-r11b-HSD1 had a lower affinity for its physiological substrate (11-dehydrocorticosterone) than did the human recombinant enzyme (KM values of 3.1 and 0.6 lM, respectively). The specific oxidoreductase activity (Vmax ) was found to be 10-fold higher for the rat than for the human enzyme (Table 2).
Fig. 3. Purification of human and rat 11b-HSD1 by cobalt affinity chromatography. (A) Purification of human Nhis-11b-HSD1 by cobalt ion affinity chromatography from P. pastoris microsomes, as outlined in Fig. 2. Lanes 1 ¼ microsomes (110 lg), 2 ¼ Triton X-100 extract (20 lg), 3 ¼ Novex Mark12 Mw marker, 4 ¼ flowthrough (0.07% v/v), 5–6 ¼ first + last wash (0.1% v/v), and 7–9 ¼ elutions (2% v/v). The samples were run on a 10% acrylamide (Bis-Tris-MOPS buffered) NuPAGE (NOVEX, Invitrogen) and subsequently stained with coomassie blue. (B) Purification of rat Nhis11b-HSD1 by cobalt ion affinity chromatography from P. pastoris microsomes, as outlined in Fig. 2. Lanes 1 ¼ microsomes (90 lg), 2–3 ¼ Triton X100 extract (20 lg), 4 ¼ Novex Mark12 Mw marker, 5 ¼ flowthrough (0.07% v/v), 6 ¼ last wash (0.1% v/v), and 7–8 ¼ elution (2% v/v). Samples were run on a 4–12% (Bis-Tris-MES buffered) NuPAGE (NOVEX) and subsequently stained with Coomassie blue.
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Discussion The principle of local inactivation and reactivation of steroids and other hormones is now well established (reviewed in [14]). The general theme of separate enzymes catalyzing the forward and reverse reactions was first established for sex hormones, which are inactivated and reactivated by separate 17b-hydroxysteroid dehydrogenases [15]. The principle of local tissue metabolism also applies to glucocorticoid hormones, where inactivation of cortisol (and corticosterone) by 11b-HSD type 2 is undisputed [1]. Originally, 11b-HSD1 was believed to be a dehydrogenase, since Lakshmi and Monder [4] could detect only dehydrogenase activity when they first purified the enzyme from rat liver in 1988. It was later demonstrated that 11b-HSD1 functions primarily as an oxidoreductase in intact cells, e.g., in the liver [16], adipocytes [17], and neurons [18]. The fact that the dehydrogenase activity of 11bHSD1 is much more stable in vitro is intriguing. Previous data suggest that factors present in the intracellular environment are important for the oxidoreductase activity. Agarwal et al. [9] found that regeneration of NADPH employing glucose-6-phosphate dehydrogenase and glucose-6-phosphate enhances the oxidoreductase activity, although this activity was still found to be less stable than the dehydrogenase activity. Detailed studies on kinetic mechanisms and posttranscriptional regulation require a highly purified enzyme. Most previous attempts to purify native 11bHSD1, e.g., from rat [4], rabbit [19], guinea pig [20], and human [21] have confirmed the instability of the oxidoreductase activity in vitro. In one study, 11b-HSD1 purified from mouse liver retained oxidoreductase activity, but the KM of this reaction was reported to be 220 lM [22]. This is surprisingly high, not only in comparison to the KM values of 0.6 and 3.1 lM obtained in the present study, for the enzymes from human and rat, respectively, but also in comparison with KM values obtained using more crude extracts [5,11,23]. In a recent study, a truncated form of human 11b-HSD1 expressed in E. coli was reported to display both dehydrogenase and oxidoreductase activities [24]. However, no details on the latter activity were given, except that the KM value was 9.5 lM. The strategy behind the purification procedure developed here was to avoid affinity chromatography using either cofactor or substrate as ligand, since previous reports indicate that no oxidoreductase activity is recovered using such ligands [4,17,18,20]. To this end, recombinant enzymes were designed to contain an affinity handle, which would enable affinity purification without disturbing the enzymatic activity. Thus, the hexa-histidine affinity handle was attached to the Nterminus of 11b-HSD1, which in vivo is situated at the
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surface of the endoplasmic membrane, opposite to that of the catalytic domain from which it is separated by a transmembrane segment containing approximately 20 amino acids [3,25]. The data presented here indicate that this affinity handle did not affect the catalytic activities of the enzymes (Table 1). Furthermore, our data strongly suggest that gentle disruption of the yeast cells is important for retention of the oxidoreductase activity. This finding might explain some of the earlier failures in obtaining active enzyme upon purification from yeast or bacteria disrupted using harsher procedures [13,26] and is in agreement with the conclusions reached by Walker et al. [24]. In addition, more oxidoreductase activity was recovered when detergent was omitted from the elution buffer employed for the affinity purification. This observation was unexpected, considering the fact that the protein contains a hydrophobic membrane anchor. Both this finding and the apparent sensitivity to sonication might be explained by dissociation of aggregates of significance for the oxidoreductase activity. For example, rat 11b-HSD1 has been shown to form aggregates of 170 or 340 kDa, with and without detergent, respectively [4]. The catalytic properties of purified human and rat 11b-HSD1 determined here were similar to those reported earlier. For example, our recombinant Nhisr11b-HSD1 displays a KM of 2.5 lM for 11-dehydrocorticosterone, which is comparable to the KM value of 1.8 lM for native rat 11b-HSD1 [4]. Furthermore, our enzyme has a KM for corticosterone of 3.1 lM, which is comparable to the corresponding value of 2.8 lM reported for recombinant rat enzyme assayed in cell extracts [5]. However, direct comparisons of values cannot be made, since different investigators have used different pH values in their assay medium. Here, a physiological pH of 7.4 was employed. In conclusion, expression of an appropriately tagged protein in yeast, followed by a simple single-step affinity purification procedure, has been successfully developed here to obtain full-length 11b-HSD1 with retention of oxidoreductase activity. This procedure is equally effective with 11b-HSD1 from both human and rat. This successful purification will facilitate future detailed biochemical and biophysical characterization of this important enzyme.
Acknowledgments ke We are grateful to Andrea Varadi, Sven-A Franzen, and Marianne Israelsson for DNA sequencing, to Margareta Forsgren for cloning of the human 11bHSD1 and for technical advice, and to Camilla Sivertsson for help with the fermentor cultures. Drs. Udo Oppermann and Joakim Nilsson are acknowledged for fruitful discussions and helpful comments on the
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manuscript. We also want to thank Professor Joe de Pierre for help with the manuscript. C.S.I. Nobel acknowledges funding for a postdoctoral position by Pharmacia & Upjohn AB and grants from the Department of Biochemistry and Biophysics at Stockholm University, Carl TryggerÕs Stiftelse, and Helge Ax:son JohnsonÕs Stiftelse.
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