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INTRODUCTION |
In mammalian tissues, two isozymes of 11-
-hydroxysteroid
dehydrogenase (11
-HSD)1
catalyze the interconversion of hormonally active
C11-hydroxylated corticosteroids (cortisol, corticosterone)
and their inactive C11-keto metabolites (cortisone,
11-dehydrocorticosterone). The 11
-HSD1 and 11
-HSD2
isozymes share only 14% identity and are separate gene products
with different physiological roles, regulation, and tissue distribution
(1). 11
-HSD2 is a high affinity NAD-dependent dehydrogenase that protects the mineralocorticoid receptor from glucocorticoid excess; mutations in the HSD11B2 gene
explain an inherited form of hypertension, the syndrome of apparent
mineralocorticoid excess in which cortisol acts as a potent
mineralocorticoid (2). By contrast, 11
-HSD1 is a relatively low
affinity NADP-dependent enzyme that acts predominantly as a
reductase in vivo, although extracted enzyme typically shows
predominant dehydrogenase activity (3). By converting cortisone to
cortisol, 11
-HSD1 facilitates glucocorticoid hormone action in key
target tissues such as liver and adipose tissue, and as such has been
implicated in a number of disorders including insulin resistance and
central obesity (4, 5).
11
-HSD1 is a member of the short chain alcohol dehydrogenase family,
also known as the short chain dehydrogenase/reductases (SDRs). SDRs
typically exhibit residue identities only at the 15-30% level,
indicative of early duplicatory origins and extensive divergence
(6-8). However, in contrast to other SDR members, 11
-HSD1 is
unusual in possessing a single transmembrane helix at the N terminus.
This is intrinsic to the endoplasmic reticulum (ER) membrane, with a
short 5-amino acid N-terminal region on the cytosolic side and the main
catalytic domain of the protein facing the lumen of the ER (9, 10). The
importance of the transmembrane domain on 11
-HSD1 activity has been
studied but with inconclusive results. An N-terminally truncated
variant of rat 11
-HSD1 was expressed in COS cells and reported to be
inactive (11, 12). However, this construct encoded a protein that had lost more than just the transmembrane helix and therefore may have lost
vital parts of the enzymatic domain. In addition, because the
expression studies were performed in COS and Chinese hamster ovary
cells, the truncated protein would have been targeted (because of the
lack of signal sequence) to the cytosol and not the ER. The lumen of
the ER promotes the formation of disulfide bonds, and studies have
indicated that there are important intrachain disulfide bonds
within the 11
-HSD1 protein (9).
The catalytic domain is glycosylated (13-15), which is in agreement
with a lumenal orientation. Experiments to resolve the importance of
glycosylation have also yielded varying results. Enzymatic
deglycosylation of rabbit (9) and human (13) 11
-HSD1 has indicated
that glycosylation is not important for enzyme activity. However,
partial inhibition of glycosylation of the rat enzyme by tunicamycin
decreased dehydrogenase activity but not reductase activity (14), and
mutation of the rat (15) and human (13) sequences at putative
N-glycosylation sites resulted in reduced or abolished activity.
Expression of human (and squirrel monkey) clones of 11
-HSD1 has been
achieved in COS cells (11, 12), HEK cells (16), and the yeast
Pichia pastoris (13, 17) using a variety of vectors. This
has led to ambiguous kinetic results with over 10-fold variation in
Km values and often significant differences in
activity between whole cells and lysates. These systems have not
yielded large amounts of pure recombinant protein, and no structural
information has come from them. Overexpression of 11
-HSD1 in
bacterial cells has been reported (17), but the resulting protein was
inactive. Failure to obtain activity was attributed to either
insolubility of the protein, and subsequent refolding problems, or a
lack of glycosylation. In this study we sought to maximize the
production of soluble recombinant human 11
-HSD1 within
Escherichia coli by varying the expression construct, the host strain, and the incubation conditions. In particular, because 11
-HSD1 is thought to contain disulfide bonds, we have assessed the
value of E. coli strains that promote disulfide bond
formation within the cytoplasm of the bacterium through mutations in
the genes encoding thioredoxin reductase and/or glutathione reductase. We also tested the effect of thioredoxin fusions, histidine tags, glycosylation status, the presence of the transmembrane domain, and
mutation of a nonconserved cysteine residue on the activity of human
11
-HSD1. Through these measures, we arrived at an optimal construct
and E. coli host combination for producing sufficient protein for purification.
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EXPERIMENTAL PROCEDURES |
Materials--
Detergents, enzyme substrates, cofactors,
chromatographic media, and chemicals were obtained from Sigma-Aldrich
(Dorset, UK) unless otherwise stated. The
[1,2,6,7-3H]cortisol (specific activity, 70.0 Ci/mmol)
was supplied by PerkinElmer Life Sciences. Oligonucleotide
primers were made by Alta Bioscience (Birmingham, UK). Restriction
endonucleases were obtained from Promega (Southampton, UK).
Production of 11-
-Hydroxysteroid Dehydrogenase 1 Expression
Vectors--
The human 11
-HSD1 cDNA (3) was subcloned into
pCDNA3.1 (16). This was further subcloned into pET21b(+) and
pET32b(+) E. coli expression vectors (Novagen). Four
distinct constructs bearing alterations to the N and C termini were
synthesized (Fig. 1). Modifications to the inserts in the pET21b(+)
vector were made to truncate the hydrophobic N terminus of 11
-HSD1,
both with (pET21CDH) and without (pET21CD) a C-terminal 6-histidine tag
(6xHis) sequence. Each of these modifications also introduced an
NheI restriction site coincident with the ATG start codon. The pET32b(+) vector was used to produce an N-terminal fusion protein
between the vector-encoded thioredoxin gene (TrxA) and either the full-length 11
-HSD1 gene (pET32FL) or an
N-terminally truncated version containing only the catalytic domain
(pET32CD). This strategy also incorporated an N-terminal 6xHis tag to
assist purification and introduced an NcoI site coincident
with the ATG start codon. The original full-length cDNA in
pCDNA3.1 was used as template for polymerase chain reaction
amplification and the forward primers (pET32CD 5', pET32FL 5', and
pET21 5'; see Table I) in conjunction
with the appropriate reverse primers (which allowed the introduction of
an XhoI restriction site downstream). The resulting
polymerase chain reaction products were subcloned into pGEM-T Easy
vector (Promega). After digestion with the appropriate restriction
enzymes, fragments were gel-purified and ligated with the appropriate
pET expression vector to give the final constructs (Fig. 1). The
direction and nucleotide sequence of the inserted cDNAs were
confirmed by sequencing. For expression studies the plasmids were
subcloned into the E. coli strains BL21(DE3), AD494(DE3), and Origami(DE3) (Novagen).
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Table I
Oligonucleotides used for the generation of expression constructs
Restriction sites are underlined and in bold face.
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Site-directed Mutagenesis--
A mutation was introduced into
the expression construct pET21CD by polymerase chain reaction using the
QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).
Primers were designed to mutate the cysteine at position 272 of the
human type 1 sequence to the corresponding residue of 11
-HSD1 from
the squirrel monkey, namely a serine (18). The oligonucleotides used
were 5'-CAGAAATCCATCCAGGAAGATC-3' and
5'-GATCTTCCTGGATGGATTTCTG-3' (the mutated nucleotides are in
boldface). The resulting construct (designated pET21CD-C272S) was verified by sequencing.
Expression of Recombinant 11
-HSD1--
Overnight cultures of
E. coli expressing the pET constructs in LB medium
containing 50 µg/ml carbenicillin (for host strain BL21(DE3)),
supplemented with 15 µg/ml kanamycin for AD494(DE3) and Origami(DE3),
and 12.5 µg/ml tetracycline for Origami(DE3) were seeded (0.1%) into
fresh LB medium containing appropriate antibiotics and then incubated
at 37 °C for 6 h (corresponding to an approximate absorbance at
600 nm of 1.0). Expression of recombinant 11
-HSD1 was then induced
by adding 1 mM
isopropyl-
-D-thiogalactopyranoside (IPTG). Control
incubations without IPTG induction were also performed. Incubation was
continued for 16 h, at 15 °C unless otherwise stated, with
shaking at 230 rpm.
Preparation of Cleared Lysates--
Bacterial cultures from
induced and uninduced E. coli cultures were pelleted by
centrifugation. The cells were disrupted by resuspension in BugBuster
reagent (Novagen) containing protease inhibitors (Mini-Complete
EDTA-free, Roche Molecular Biochemicals) and benzonase DNase (Novagen)
using 50 µl of lysis reagent/ml of original culture. For fuller lysis
of the cells, lysozyme was included to a final concentration of 200 µg/ml. After incubation at room temperature with shaking for 25-30
min, the cell debris was pelleted by centrifugation at 11,000 × g for 10 min. The supernatant was removed for activity
assays and protein determination (Bio-Rad protein reagent).
Activity Assays--
Cleared lysates and other enzyme
preparations (typically 1-10 µl) were incubated in 0.5 ml of
phosphate buffer (0.1 M, pH 7.6) containing 50,000 cpm
[3H]cortisol, 100 nM unlabeled cortisol, and
200 µM NADP for 30 min at 37 °C to assess
dehydrogenase activity or [3H]cortisone (generated as
reported previously (19)), 100 nM unlabeled cortisone, 200 µM NADPH, and a regeneration system (10 mM
MgCl2, 5 mM glucose-6-phosphate, and 10 units
glucose-6-phosphate dehydrogenase) (20) to assess reductase activity.
Steroids were partitioned into 10 volumes of dichloromethane and
separated by TLC using ethanol/chloroform (8:92) as the mobile phase.
The TLC plates were analyzed on a Bioscan radioimaging detector, and
the fractional conversion of cortisol to cortisone or cortisone to cortisol was used to estimate enzyme activities.
Activity was also assessed in intact E. coli cells.
Bacterial cultures (1 ml) were centrifuged, and the resulting pellet
was resuspended in 0.5 ml of phosphate buffer (0.1 M, pH
7.6) containing either 100 nM cortisol plus
[3H]cortisol tracer, to assess the levels of
dehydrogenase activity, or 0.5 ml of phosphate buffer (0.1 M, pH 7.6) containing 100 nM cortisone and
[3H]cortisone tracer, to measure reductase activity.
Kinetic Analysis--
Enzyme activities were assayed in the
standard reaction mixture containing cleared lysates or purified
protein, the appropriate cofactor (NADP or NADPH plus the regeneration
system), and varying substrate concentrations (0.25-60
µM). In each case, linearity of enzyme activity
versus time was ensured. Km value estimations were averaged from Lineweaver-Burke plots derived from
three experiments as previously reported (21).
Western Blot Analysis--
SDS-PAGE was performed using the
Laemmli method (22) with 12.5% acrylamide minigels using a Bio-Rad
Mini-Protean II apparatus. 10 µg of protein from bacterial
cleared lysates, human liver homogenates, or mouse liver homogenates
(produced as reported previously (23, 24)) were loaded either in sample
buffer containing
-mercaptoethanol, to completely reduce any
disulfide bonds, or in sample buffer without
-mercaptoethanol, to
retain the disulfide bonds. Gels were stained with Coomassie
Brilliant Blue (R-250) to investigate the purity and amount
of protein in the extracts. Western blotting was performed as reported
previously (23). Briefly, after electrophoresis, the proteins were
transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA).
Nonspecific protein binding was blocked by incubating the membranes in
20% nonfat milk and 0.1% Tween 20 in phosphate-buffered saline at
25 °C for 1 h. The membranes were then incubated with a
validated polyclonal antibody to human 11
-HSD1 (The Binding Site,
Birmingham, UK) at a dilution of 1:1000 for 16 h at 4 °C. After
three 10-min washes in phosphate-buffered saline/0.1% Tween 20, the membranes were incubated with a secondary antibody (goat anti-sheep
IgG peroxidase conjugate (The Binding Site)) at a dilution of 1:75,000
for 1.5 h at room temperature. Bound peroxidase-conjugated Ig was
visualized using an ECL detection kit (Amersham Pharmacia Biotech) by
exposing the membranes to x-ray film (Kodak). Relative band intensities
were analyzed by laser scanning densitometry.
Purification of Recombinant 11
-HSD1--
A further construct
was designed for the purification of recombinant 11
-HSD. The plasmid
pET21CD was digested with NcoI and XhoI to
release the 11
-HSD insert and ligated to the similarly digested
pET28b(+) vector (Novagen). This gave the construct pET28HCD, which
contained the catalytic domain of 11
-HSD with an N-terminal 6xHis
tag to aid purification. For expression, the construct was subcloned
into E. coli strain BL21(DE3) and induced as described above. Cleared lysate (8 ml) was prepared from 200 ml of induced culture and mixed with 0.3 ml of nickel-nitrilotriacetic acid His-bind
resin (Novagen) followed by gentle shaking at room temperature for 30 min. The mixture was loaded into an empty column, and unbound protein
was removed by washing with 8 ml of 50 mM NaPO4
buffer, pH 8, containing 300 mM NaCl. Bound protein was
eluted in a stepwise fashion with the same buffer containing increasing
concentrations of imidazole (2 × 0.2 ml each of 40, 60, and 100 mM imidazole). The eluted fractions were tested for enzyme
activity, and the purity was assessed by SDS-PAGE.
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RESULTS |
Expression of Different Variants of 11-
-Hydroxysteroid
Dehydrogenase 1 in E. coli--
Four constructs were designed (Fig.
1) to test the effect of (a)
removing the N-terminal transmembrane region of 11
-HSD1, (b) including a 6xHis tag at the C terminus, and
(c) fusing the N terminus of the catalytic domain to
thioredoxin, a procedure reported to increase the solubility of
recombinant proteins (25), particularly those requiring disulfide
bonds. Initial comparisons used a thioredoxin reductase-deficient
strain of E. coli, AD494(DE3), that has been reported to
enhance the formation of disulphide bonds within the bacterial
cytoplasm (26), particularly for thioredoxin fusion proteins. In
addition, because initial tests indicated that very little soluble
protein was produced by incubation entirely at 37 °C, cultures were
switched to 15 °C after the addition of IPTG to increase the
possibility of producing soluble protein (26). The overall level of
expression from each of the four constructs under these conditions was
estimated using SDS-PAGE gels and subsequent densitometric analysis.
All constructs produced protein of the expected size, with levels
varying from 15% of the total cell protein for the thioredoxin
full-length 11
-HSD1 fusion (pET32FL) to 35% for the nontagged
catalytic domain (pET21CD).

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Fig. 1.
Diagrammatic representation of
11 -HSD1 expression constructs.
Clear boxes represent the pET32 vector-encoded thioredoxin
gene (TrxA), and the 6xHis-tag sequences are denoted by
black boxes. The diamond hatched region is the
N-terminal region that includes not only the transmembrane domain but
also the 5/6 amino acid extreme N terminus (amino acid sequence shown
above). The diagonally hatched boxes represent
the catalytic domain of 11 -HSD1 spanning amino acid residues
24-287. Additional amino acids (MAS), produced as a
consequence of cloning, are shown by their single-letter codes at the N
terminus of the pET21 construct. A mutation was made in the pET21CD
construct at amino acid position 272, changing a cysteine residue to a
serine, and is highlighted above.
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The extraction of proteins by sonication indicated that some of the
recombinant protein was in a soluble form (data not shown). Despite
this, no enzyme activity could be detected in the sonicated lysates.
However, incubation of intact bacterial cells (transformed with the
pET32CD construct) with labeled cortisol and cortisone indicated that
the bacteria had acquired both 11-
-hydroxysteroid dehydrogenase and 11-oxo reductase activities (Fig.
2). This was specific to IPTG-induced
cultures and hence indicative of the presence of active recombinant
enzyme within the cells prior to sonication. Moreover, a gentler lysis
of the cells by a mixture of a commercial detergent (BugBuster) and
lysozyme produced an extract with clear 11
-HSD activity for some of
the constructs (Fig. 3). Examination of
these extracts and the remaining pellets by SDS-PAGE indicated that all
the constructs were expressing protein that could be detected easily in
the pellets (Fig. 3B) but was not easily visible on
Coomassie staining of the supernatants (Fig. 3A). However,
Western immunoblots of the supernatant samples using a specific
antiserum to 11
-HSD1 clearly showed that soluble recombinant protein
was being expressed for at least three of the constructs (Fig.
3C), with the order of expression being pET21CD (catalytic
domain alone) > pET32CD (thioredoxin catalytic domain fusion) > pET21CDH (catalytic domain + His tag). No
expression was observed with the pET32FL (thioredoxin full-length
fusion) construct. Production of active recombinant 11
-HSD1 was
assessed by measuring the ability of cleared lysates to convert
cortisol to cortisone (11-
-hydroxysteroid dehydrogenase reaction).
Comparison of these enzyme activities (Fig. 3D) indicated
that the construct encoding the catalytic domain alone (pET21CD)
produced the highest levels of activity, concomitant with the highest
levels of protein observed on the Western blots. Interestingly, for
this construct the regulation of protein expression was poor, with
similar protein and enzyme activity being observed in the presence and
absence of IPTG. Fusion of the catalytic domain with thioredoxin
(pET32CD), which included an intervening N-terminal 6xHis tag, also
resulted in active soluble protein, albeit at a lower level than that
seen with the catalytic domain alone. However, the addition of a
C-terminal 6xHis tag (pET21CDH) resulted in almost no activity despite
soluble protein being evident on Western blot analysis. Inclusion of
the transmembrane domain in the thioredoxin fusion (pET32FL) resulted in no enzyme activity, which is in agreement with a lack of detectable protein on the blots. Additional constructs were produced that encoded
the full-length 11
-HSD1 sequence in pET21b(+) (i.e. not as a fusion), but these constructs repeatedly failed to express any
recombinant protein in either a soluble or insoluble form (data not
shown).

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Fig. 2.
Representative thin layer radiochromatograms
showing the enzyme activity of intact E. coli cells
containing the 11 -HSD1 expression construct
pET32CD. A, the conversion of 100 nM cortisol
(F) to cortisone (E). B, the
conversion of 100 nM cortisone to cortisol. The top
panels in each case represent activity observed from uninduced
control cultures.
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Fig. 3.
Analysis of 11 -HSD1
expression constructs. A, Coomassie Blue-stained
SDS-PAGE gel of the supernatant fractions of 11 -HSD1 expression
constructs. No clear recombinant protein bands could be detected.
B, Coomassie Blue-stained SDS-PAGE gel of the pellet
fractions of 11 -HSD1 expression constructs. Recombinant 11 -HSD1
from pET21 constructs is clearly visible at ~29 kDa (pET21-CD) and 30 kDa (pET21-CDH), whereas those for the pET32 constructs are at 45 kDa
(pET32-CD) and 47 kDa (pET32-FL) because of the presence of the
TrxA gene. C, Western blot of the supernatant
fractions of 11 -HSD1 expression constructs showing soluble protein
was produced from pET21CD, pET21CDH, and pET32CD. D,
activity data (cortisol to cortisone) for the 11 -HSD1
expression constructs showing highest levels of activity for pET21CD.
No activity was obtained from pET32FL, which is coincident with no
protein expression.
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Expression of 11-
-Hydroxysteroid Dehydrogenase 1 in Different
Strains of E. coli--
The two constructs that demonstrated enzyme
activity (pET21CD and pET32CD) were then used to compare the effect of
E. coli host strain on the production of active protein,
with the particular purpose of comparing strains reputed to enhance
disulfide bond formation by mutations in either thioredoxin reductase
or glutathione reductase genes (26). Thus the thioredoxin
reductase-deficient strain, AD494(DE3), was compared with the
thioredoxin reductase- and glutathione reductase-deficient strain
Origami(DE3) and a strain deficient in neither enzyme, BL21(DE3).
Surprisingly, for the pET21CD construct, both activity assays (Fig.
4C) and Western analyses (Fig.
4B) clearly indicated that BL21(DE3) cells gave better
protein expression and activity than AD494(DE3) or Origami(DE3), with
the latter giving negligible levels of both protein and activity. As
before (Fig. 3), the thioredoxin fusion construct pET32CD yielded lower
amounts of protein and enzyme activity compared with the nonfusion
construct pET21CD, although in this case the host strain had much less
effect on the level of expression (Fig. 4), with all three strains
giving similar results. Examination of Coomassie-stained SDS-PAGE gels
indicated that the levels of soluble recombinant proteins in the
lysates were very low (Fig. 4, A and B), with only faint bands being evident in the highest expressing combinations of construct and host strain. Using E. coli strains
BL21(DE3) and AD494(DE3), a comparison was made between protein
extraction produced with detergent (BugBuster) alone and detergent with
the addition of lysozyme, the latter combination having been shown microscopically to cause complete lysis of the cells and to release approximately twice the amount of protein from the bacterial cells when
compared with detergent alone (data not shown). This analysis showed
that almost all the active 11
-HSD1 could be released by detergent
alone, without lysozyme, resulting in a preparation in which high
enzyme activity was accompanied by a clear band on Coomassie-stained
gels (Fig. 5).

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Fig. 4.
Expression of
11 -HSD1 in different E. coli
host strains. A, Coomassie Blue-stained SDS-PAGE gel
of the supernatant fractions of 11 -HSD1 expression constructs from
different host strains. B, Western blot of the supernatant
fractions. The highest expression for the pET21CD construct was
obtained in the BL21(DE3) host strain > AD494(DE3). No
recombinant protein was produced in Origami cells using this construct.
Expression levels for pET32CD were similar in all three host strains.
C, activity data (cortisol to cortisone) for the 11 -HSD1
expression constructs showing greatest activity from the pET21CD
recombinant protein in combination with BL21(DE3).
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Fig. 5.
Effect of lysis reagent on amount and
activity of recombinant of 11 -HSD1 in
different E. coli host strains. A,
Coomassie Blue-stained SDS-PAGE gel of supernatant fractions of
11 -HSD1 protein extracted with (left hand panel) and
without (right hand panel) lysozyme. Protein bands
(indicated by the arrow) could be identified from the
cleared lysates produced without the addition of lysozyme.
B, Western blot of the extracted proteins. C,
activity data (cortisol to cortisone) for the extracted protein.
Equivalent levels of activity were obtained from cleared lysates
independent of the addition of lysozyme.
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Mutational Analysis of the Enzyme--
Because human 11
-HSD1
contains four cysteine residues, three of which are conserved across
all mammalian 11
-HSD1 proteins, we tested for the presence of
interchain disulfide bonds by probing Western blots of SDS-PAGE gels of
lysates run under both reducing and nonreducing conditions (Fig.
6A). The results clearly
showed the presence of both dimer and monomer bands in the nonreducing lanes, suggesting that some of the protein existed in an interchain disulfide-bonded dimeric form. Examination of human liver extracts indicated that the natural enzyme also consisted of a similar combination of monomeric and dimeric forms (Fig. 6B),
although analysis of mouse liver extracts (Fig. 6C) showed
the presence of only monomers. Because the human 11
-HSD1 contains an
additional cysteine (Cys-272) when compared with the other mammalian
sequences reported to date, we investigated the effect of mutating this residue to the corresponding residue of 11
-HSD1 from squirrel monkey, namely a serine (18). Interestingly, the expression of this
mutant in E. coli produced very similar dehydrogenase and
reductase activities to those observed with the wild type and no
significant alteration in the respective Km values. However, Western blots of nonreducing gels indicated that the ability
of the protein to form disulfide-bonded dimers had been abolished
(Fig. 6D).

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Fig. 6.
Western analysis of SDS-PAGE gels under
reducing (+ -mercaptoethanol) and nonreducing
( -mercaptoethanol) conditions.
A, cleared lysates of pET21CD showing monomer (molecular
mass, 29 kDa) and dimer (58 kDa) species of 11 -HSD1 under
nonreducing conditions. B, human liver extracts showing
dimeric (68 kDa) and monomeric (34 kDa) forms of 11 -HSD1.
C, mouse liver extracts showing the presence of monomeric
11 -HSD1 only (32 kDa). D, cleared lysates from
pET21CD-C272S showing monomeric 11 -HSD1 and the loss of the
interchain disulfide-bonded dimeric forms.
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Purification of Recombinant 11
-HSD1--
Several attempts at
purification of recombinant pET21CD from BL21(DE3) cells were
unsuccessful. Several combinations of gel filtration, ion exchange, and
ADP-agarose methods failed to yield sufficiently pure protein. Because
the addition of sequences at the N terminus of the catalytic domain of
11
-HSD did not seem to affect activity, a further expression
construct based on our most active pET21CD plasmid was generated that
incorporated an N-terminal 6xHis tag to allow purification by metal
affinity chromatography. Use of this construct (pET28HCD) in BL21(DE3)
cells resulted in lysates from which the enzyme could be purified to
apparent homogeneity, as indicated by SDS-PAGE, in a single
chromatographic step (Fig. 7). Activity
measurements indicated that the recombinant human 11
-HSD1 had
been purified 159-fold with an overall yield of 28% (Table
II). The final specific activity of the
purified enzyme was 42.47 pmol/h/µg of protein. The purified enzyme
had activity in both dehydrogenase (cortisol to cortisone) and
reductase (cortisone to cortisol) directions, with the estimated
Km for cortisol being 1.4 µM (± 0.6 S.D.) and for cortisone being 9.5 µM (± 0.9 S.D.), both
values being similar to those reported from mammalian systems (reviewed
in Ref. 1).

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Fig. 7.
Gel electrophoresis of the purification steps
for recombinant 11 -HSD1. Coomassie
Blue-stained SDS-PAGE gel of various fractions from the purification of
recombinant protein by His-bind affinity chromatography. Lane
1, initial cleared lysate from an induced culture of BL21(DE3)
cells containing pET28HCD; lane 2, fraction eluted from the
column with 40 mM imidazole; lane 3, fraction
eluted with 60 mM imidazole; lane 4, final
eluate using 100 mM imidazole.
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DISCUSSION |
11-
-Hydroxysteroid dehydrogenase activity was first documented
in the rat liver in the 1950s, but it was the studies of Tannin et al. (3) that resulted in the enzymatic characterization, purification, and cloning of the liver-type 11
-HSD isozyme. With the
cloning of a second "kidney-type" 11
-HSD isozyme, the liver-type isozyme is now termed 11
-HSD1.
The importance of these isozymes in the metabolism and clearance of
glucocorticoids is well established; in addition, these enzymes are
intricately involved in the pathogenesis of human diseases. For
example, 11
-HSD2 is implicated in hypertension and fetal growth
retardation (1). Specifically, for 11
-HSD1, emerging data have
highlighted the role of this enzyme in modulating insulin sensitivity
and visceral adiposity. Thus mice lacking the HSD11B1
gene are resistant to hyperglycemia of stress/feeding because of a failure to activate glucocorticoid within the liver and
stimulate gluconeogenesis (4). Improvements in insulin sensitivity in
normal volunteers given the 11
-HSD1 inhibitor carbenoxolone support
such a concept (27). Similarly in visceral adipose tissue, 11
-HSD1
acts locally to generate active glucocorticoid concentrations, thereby
stimulating adipogenesis (5, 28). Defect in the activity of 11
-HSD1
is also thought to underpin an inherited form of polycystic ovary
syndrome, the syndrome of apparent cortisone reductase deficiency (29).
Finally, further studies are investigating the role of the enzyme in
central nervous system tissues and its relationship to
neurodegenerative diseases (30). In each case though, the exciting
concept has emerged that modulation of 11
-HSD1 expression may
represent a novel mechanism to modulate glucocorticoid action at the
tissue level without changing circulating concentrations, thereby
precipitating states of glucocorticoid excess or deficiency. Thus there
is a clear clinical need to undertake a detailed characterization of
the human 11
-HSD1 isozyme with a view to its purification.
11
-HSD1 belongs to the SDR superfamily, as defined on the basis of
an N-terminal nucleotide binding motif, a central active site, and
consensus sequence data. Sophisticated analytical approaches suggest
that there are over 1000 members of this superfamily (6-8), with only
one residue (Tyr) being strictly conserved. A lysine 4 residues
downstream and a serine 14 residues upstream are also largely
conserved; all these residues are present in 11
-HSD1. A model for
catalysis of SDRs has been proposed on the basis of these residues
(31). Binding of the coenzyme, NAD(H) or NADP(H), is in the N-terminal
part of the molecule involving a common protein folding arrangement of
- and
-strands ("Rossmann" fold) associated with a common
Gly(Xaa)3GlyXaaGly motif (also found in 11
-HSD1). The
critically important tyrosine seems to maintain a fixed position relative to the scaffolding of the Rossmann fold and the cofactor position, whereas the substrate-binding pocket alters in such a way
that the dehydrogenation/reduction reaction site is brought into
bonding distance of the tyrosine hydroxyl group. The tyrosine therefore
acts as a basic catalyst, the lysine binds to NAD/P(H) and lowers the
pKa value of the tyrosine, and the serine plays a
subsidiary role of stabilizing substrate binding (31, 32).
Several groups have evaluated the importance of the N-terminal domain
of 11
-HSD1. Recently it has been shown that the orientation of the enzyme within the ER is determined by sequences close to the N
terminus (10). Chimeric proteins where the N-terminal regions,
including the membrane anchors, of the 11
-HSD1 and 11
-HSD2 enzymes were exchanged adopted inverted orientations in the ER membrane
(10). Neither protein was catalytically active. However, mutation of a
single lysine residue close to the N terminus of type 1 resulted in an
inverted orientation without loss of activity. These results suggest
that the N-terminal anchor is required for both activity and correct
orientation, although it should be noted that the sequences exchanged
in the chimeras included much more than just the transmembrane helix.
Mercer et al. (12) reported that expression of an
N-terminally truncated 11
-HSD1 did not produce a soluble protein.
However, these studies and others have employed mammalian expression
systems where such constructs would not be appropriately targeted to
the ER, and hence correct folding and disulfide bond formation may not
have been facilitated.
In this study, using a series of bacterial expression constructs, we
have shown that the activity of human 11
-HSD1 does not depend on the
N-terminal domain. Constructs where the N-terminal region had been
removed (pET32CD and pET21CD) exhibited higher levels of expression and
activity than constructs containing the entire 11
-HSD1 sequence.
Moreover, inclusion of the transmembrane domain, either with or without
the thioredoxin fusion partner, failed to produce soluble active
protein. This is in agreement with a study carried out by Blum et
al. (13), in which the complete human 11
-HSD1 sequence was
expressed in E. coli and resulted in a protein that was
virtually insoluble, difficult to purify, and completely inactive.
We also investigated expression systems in which thioredoxin, the
product of the E. coli TrxA gene (25), was a fusion partner. In many cases heterologous proteins produced as thioredoxin fusion proteins are correctly folded and display full biological activity (25,
33-35). This has been thought to be caused by the small, highly
soluble nature of thioredoxin, which also has robust folding characteristics (36). However, in our study proteins produced as a
fusion with thioredoxin at the N terminus (pET32CD and pET32FL) showed
no overall increase in the levels of soluble protein when compared with
nonfusion constructs (pET21CD and pET21CDH), indicating that such
fusions are not always profitable. Similarly, fusion of a 6xHis tag at
the C terminus, as a means to simplify purification, was also
detrimental to the solubility, and particularly the activity, of the
enzyme. Residues close to the C terminus of SDRs may frequently be
important in substrate binding (37), and modifications in this region
may thus affect protein structure to the detriment of enzyme activity.
This study also clearly resolves the issue of whether glycosylation is
required for the activity of the human enzyme. Studies on the rat
11
-HSD1 enzyme indicated that partial inhibition of glycosylation
with tunicamycin inhibited dehydrogenase activity by 50% but had no
effect on reductase activity (14). Mutagenesis of the first of two
potential N-glycosylation sites reduced dehydrogenase and
reductase activities by 75 and 50%, respectively, whereas mutagenesis
of the second site completely abolished activity (15). Conversely,
studies carried out on the rabbit enzyme, which like the human
homologue contains three potential glycosylation sites, suggest that
glycosylation is not important for enzyme activity. No alteration in
activity could be observed after complete deglycosylation of rabbit
11
-HSD1 (9). Conflicting studies on the human enzyme have also been
reported. Recently, human 11
-HSD1 has been expressed in E. coli, where the biosynthesis of N-linked glycoproteins
does not occur. This resulted in a recombinant protein that was
completely devoid of enzyme activity (17). The same group also
investigated the effects of deglycosylation on human 11
-HSD1
purified from liver and recombinant protein produced by the yeast
P. pastoris (13). Site-directed mutagenesis of the three
potential glycosylation sites yielded an inactive protein from yeast
cells as assessed using metyrapone and metyrapol as the substrates.
However, the enzyme purified from human liver, upon complete
deglycosylation, remained fully active. The results here agree with the
latter experiment and clearly show that nonglycosylated enzymatically active 11
-HSD1 can be generated within E. coli, with the
recombinant enzyme possessing both reductase and dehydrogenase
activities with similar kinetic properties to those reported previously
from mammalian expression systems. Glycosylation is therefore not
required for activity or protein folding, although it could still be
important for protein stability within the endoplasmic reticulum.
All the constructs used in this study gave only moderate levels of
soluble protein but a high proportion of protein in an insoluble
form. The lack of protein solubility in E. coli is a complex event with many contributing factors. Although fusion with
heterologous proteins may sometimes help to redress many solubility
problems, another factor that may be important is the inability of the
recombinant protein to form key disulfide bonds in the reducing
environment of the bacterial cytoplasm (38). Rabbit 11
-HSD1 is known
to contain an intrachain disulfide bond (9), and therefore we
investigated the expression levels and activity of our recombinant
proteins in a variety of host E. coli strains, some of which
have been developed to promote disulfide bond formation. Within
E. coli at least two systems are responsible for reducing
disulfide bonds that form in the cytoplasm; the thioredoxin system that
consists of thioredoxin reductase and thioredoxin and the glutaredoxin
system that includes glutathione reductase, glutathione, and
glutaredoxins. We evaluated this using three separate E. coli strains. It was anticipated that disulfide bonds, and
therefore solubility and activity of the soluble protein, would be
enhanced by the use of AD494(DE3) and particularly the Origami(DE3)
strain. In effect, the reverse was observed with the highest levels of
soluble protein and enzyme activity being observed using BL21(DE3) as
the host strain. This result could imply that the intramolecular
disulfide bond observed in the rabbit 11
-HSD1 protein (9) is not
present in the human enzyme, although this awaits experimental confirmation.
We also tested for the presence of interchain disulfide bonds by
probing Western blots of SDS-PAGE gels of bacterial lysates run under
both reducing and nonreducing conditions. Both dimer and monomer bands
were identified in the nonreducing lanes, suggesting that some of the
recombinant protein exists in an interchain disulfide-bonded form.
Examination of human liver extracts indicated that the native 11
-HSD1 enzyme existed in a similar combination of monomeric and
dimeric forms, proving that the heterogeneity was not a consequence of
expression in the bacterial system. However, this heterogeneity not
only complicates the purification of 11
-HSD1, as has been noted
previously for native enzyme (9), but could also hinder crystallographic analysis because crystal growth requires pure protein
in a homogeneous form. Tests on extracts of mouse liver, however,
detected no intermolecular disulfide bridges. Because previous reports
also suggested that rabbit 11
-HSD1 contains no intermolecular
disulfide bonds (9), we investigated the effect of mutating the
additional cysteine (Cys-272), found only in the human sequence, to the
corresponding residue from the most closely related 11
-HSD1 sequence
(squirrel monkey). Expression of the resulting mutant (pET21CD-C272S)
in the optimized bacterial expression system resulted in a protein with
kinetic properties indistinguishable from the wild-type recombinant
protein. However, nonreducing gels showed that the ability to form the
interchain disulfide bonds had been abolished. Previous structural
studies on other SDRs suggest that they exist naturally as
nondisulfide-bonded dimers or tetramers (reviewed in Ref. 37). The
results here suggest that Cys-272 of human 11
-HSD1 may be involved
in disulfide bonds between adjacent polypeptide chains of the enzyme,
possibly stabilizing the dimeric form. However, this property does not seem to be vital to the activity of the enzyme.
Using a modified expression construct that included an N-terminal 6xHis
tag, we developed a simple purification protocol that allowed 157-fold
purification of recombinant human 11
-HSD1 in one step from crude
cell lysates. The purified protein demonstrated activity in both
dehydrogenase and reductase directions with Km values of 1.4 µM for cortisol and 9.5 µM
for cortisone.
In conclusion this study has shown that, despite reports to the
contrary, bacterial expression systems have the potential to produce
active soluble 11
-HSD1 protein. The results also demonstrate conclusively that the N-terminal region, containing the transmembrane domain, glycosylation, and interchain disulfide bonds are not essential
for the activity of this enzyme. For the first time, active soluble
11
-HSD1 has been produced in vitro and purified to
apparent homogeneity. This will now allow detailed functional analysis
of the enzyme using E. coli-produced protein and facilitate future structure/crystallographic studies.