Tài liệu Báo cáo khoa học: Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone docx

Production of biologically active forms of recombinant
hepcidin, the iron-regulatory hormone
Bruno Gagliardo
1
, Audrey Faye
2,3
, Maryse Jaouen
1
, Jean-Christophe Deschemin
2,3
, Franc¸ois
Canonne-Hergaux
4
, Sophie Vaulont
2,3
and Marie-Agne
`
s Sari
1
1 CNRS (UMR 8601), Universite
´
Paris Descartes, France
2 Institut Cochin CNRS (UMR 8104), Universite
´
Paris Descartes, France
3 Inserm (U567), Universite
´
Paris Descartes, France
4 CNRS (ICSN), Gif-Sur-Yvette, France
Hepcidin is a 25 amino acid, cysteine-rich peptide, first
identified in human blood [1] and urine [2] as an anti-

microbial peptide of the defensin family. Human hep-
cidin is the product of the HAMP gene, which encodes
an 84 amino acid precursor protein [3,4]. In addition,
hepcidin genes have also been identified in several
species, including the mouse [5], rat [6], pig [7], dog [8]
and fish [9–11]. In mammals, the hepcidin gene is
expressed predominantly in hepatocytes and constitutes
the master regulator of iron homeostasis. Hepcidin
gene expression is positively regulated by iron and
inflammation and negatively regulated by anemia and
hypoxia [5,12]. There is compelling evidence that
dysregulation of hepcidin underlies many iron disorders.
Most of the iron overload syndromes (primary hemo-
chromatosis and secondary iron overloads) imply a
reduction of hepcidin secretion whereas, in contrast,
overexpression of hepcidin appears to play a deter-
mining role in anemia of inflammation or inflammation
of chronic disease [13] and iron-refractory iron
deficiency anemia [14].
HAMP and Hepc1 encode precursors of 84 and 83
amino acids, respectively, which contain typical 24 and
23 amino acid endoplasmic reticulum targeting signal
motifs at the N-terminus of the human and mouse
precursors, respectively. This signal is cleaved to
produce prohepcidin, harboring a 35 amino acid
proregion and the C-terminus 25 amino acid mature
Keywords
antimicrobial peptide; Escherichia coli
expression; ferroportin; hepcidin; iron
homeostasis

Correspondence
M A. Sari, UMR 8601, Universite
´
Paris
Descartes, 45 rue des Saints-Pe
`
res, 75006
Paris, France
Fax: +33 1428 68387
Tel: +33 1428 62142
E-mail:
(Received 1 April 2008, revised 22 May
2008, accepted 28 May 2008)
doi:10.1111/j.1742-4658.2008.06525.x
Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the
central regulator of body iron metabolism. Hepcidin is synthesized under
the form of a precursor, prohepcidin, which is processed to produce the
biologically active mature 25 amino acid peptide. This peptide is secreted
and acts by controlling the concentration of the membrane iron exporter
ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to
ferroportin, inducing its internalization and degradation, thus regulating
the export of iron from cells to plasma. The aim of the present study was
to develop a novel method to produce human and mouse recombinant hep-
cidins, and to compare their biological activity towards their natural recep-
tor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin
fusion proteins. The corresponding peptides, purified after cleavage from
thioredoxin, were properly folded and contained the expected four-disulfide
bridges without the need of any renaturation or oxidation steps. Human
and mouse hepcidins were found to be biologically active, promoting ferro-
portin degradation in macrophages. Importantly, biologically inactive

aggregated forms of hepcidin were observed depending on purification
and storage conditions, but such forms were unrelated to disulfide bridge
formation.
Abbreviations
huhepc, human hepcidin encoded by HAMP gene with an extra N-terminal methionine; m1hepc, mouse hepcidin encoded by Hepc1 gene
with an extra N-terminal methionine; MES, 2(N-morpholino)ethanesulfonic acid; SELDI-TOF, surface enhanced laser desorption ionization
time of flight; TFA, trifluroacetic acid; TRX, thioredoxin.
FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS 3793
peptide. This prohepcidin is further processed through a
furin-type propeptide cleavage site to generate the
secreted 25 amino acid hepcidin [2,15]. The 25 amino
acid peptide forms a hairpin loop stabilized by four
disulfide bonds, one of which is an unusual vicinal bond
between adjacent cysteines at the hairpin turn [16]. A
recent structure ⁄ function study revealed that the
N-terminus of hepcidin is essential for its bioactivity, and
that the structure is otherwise permissive for changes [17].
Once in the circulation, the peptide acts to limit
gastrointestinal iron absorption and serum iron levels
by inhibiting dietary intestinal iron absorption and
iron recycling by the macrophages [18]. This is
achieved by hepcidin binding to ferroportin, the trans-
membrane iron transporter necessary for iron transfer
out of intestinal epithelial cells and macrophages [19],
resulting in its internalization and subsequent degrada-
tion [20,21].
Although the functional role of hepcidin in iron
metabolism has been well documented, little is known
concerning its cell biology. Chemical synthesis of
human hepcidin has been successful but difficult to

achieve [22–24] due to folding restrictions imposed by
the four-disulfide bridges [10,16]. Other studies have
reported the purification of human recombinant hepci-
din in the form of fusion proteins [25–28] and, until
very recently [29], none of the recombinant hepcidins
were shown to be bioactive in iron metabolism.
In the present study, we describe an efficient proce-
dure for purification of biologically active recombinant
hepcidins. This constitutes an important step that will
allow for a better understanding of hepcidin biology,
which is a prerequisite for the use of hepcidin in medi-
cal applications.
Results
Strategies for hepcidin expression and
nomenclature
We were able to produce properly folded recombinant
hepcidins with the correct four-disulfide bridges with-
out the need of a denaturation ⁄ renaturation step. A
Novagen pET32-LIC vector expression system was
used into which the hepcidin encoding sequence was
cloned downstream of a thioredoxin (TRX) sequence
to produce a fusion protein under the control of a T7
promoter. TRX was chosen as the fusion protein
because it is involved in disulfide bridge formation
[30,31]. A double tag system links TRX and hepcidin: a
his-tag upstream of a thrombin cleavage site and a
S-tag upstream of an enterokinase cleavage site (Fig. 1).
Noteworthy, the recombinant hepcidin produced after
cleavage exhibited an extra methionine at the N-termi-
nus compared to the native peptide, as a result of the

nature of the enterokinase cleavage site (Table 1). The
major expected characteristics of the cleaved pro-
teins ⁄ peptides are presented in Fig. 1, together with the
nomenclature used in the present study.
Origami B cells were chosen as a host strain because
they carry mutations both in TRX and glutathione
reductase genes, hence increasing cytosolic formation
of disulfide bridges compared to those obtained in the
highly reductive environment of a regular Escherichia
coli strain. This strain choice was indeed important
because mouse hepcidin encoded by Hepc1 gene with
an extra N-terminal methionine (m1hepc) purified
from a BL21(DE3) strain was found mostly as an
insoluble multimer peptide (data not shown).
Fig. 1. Scheme of hepcidin production and their expected characteristics. The name of each construct used through the study is given next
to its corresponding cartoon, together with the amino acid numbers (AA), isoelectric point and the theoretical average and monoisotopic (in
parenthesis) mass obtained using Protein Parameter software (ExPasy; Yields are expressed in %
(nmol of purified protein X100 per nmol of TRX-hepc) and are representative of three independent preparations.
Active recombinant hepcidin B. Gagliardo et al.
3794 FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS
Plasmid construction
Cloning of m1hepc and human hepcidin encoded by
HAMP gene with an extra N-terminal methionine
(huhepc) was performed using synthetic oligonucleo-
tides. Complementary hepcidin oligonucleotides were
annealed and directly cloned into the pET32LIC vector
(Table 1). Upon transformation and amplification in
E. coli TG1 cells, pET32LIC-m1hepc and pET32LIC-
huhepc vectors were purified, checked by sequencing
and used to transform Origami B cells.

Fusion protein expression
TRX-hepcidin 20 kDa fusion proteins (TRX-m1hepc
and TRX-huhepc) were produced under the same
conditions. Therefore, only TRX-huhepc production is
described here. Transformed pET32LIC-huhepc
Origami cells were cultured in high density ZYM-5052
medium at 25 °C, as described previously [32], except
that the autoinduction lactose step was replaced by a
‘classical’ isopropyl thio-b-d-galactoside induction step
after 30 h of growth (see Experimental procedures).
The decrease of temperature from 37 °Cto25°C was
found necessary to avoid the formation of inclusion
bodies because TRX-huhepc found in these structures
was highly aggregated and very difficult to recover.
Human hepcidin purification
Hepcidin purification was carried out in three steps:
(a) TRX-huhepc purification; (b) thrombin cleavage of
TRX-huhepc to generate S-huhepc; and (c) S-huhepc
enterokinase digestion to generate huhepc. For
unknown reasons, TRX-huhepc (as well as TRX-
m1hepc) appeared to be resistant to enterokinase
digestion, avoiding a direct purification of huhepc
from TRX-huhepc. The purification was followed by
SDS ⁄ PAGE, for proteins or peptides, and also
Experion
TM
capillary electrophoresis for proteins.
Step1: purification of TRX-huhepc
After 30 h of growth, 3 h of induction and lysis, 8.5 g
of soluble proteins were obtained; 8% of which com-

prised TRX-huhepc (approximately 700 mg) (Fig. 2,
lane 2). Some TRX-huhepc remained in the insoluble
fraction (Fig. 2, lane 1) and could not be recovered,
even in the presence of urea or guanidine (data not
shown). Lowering the temperature to 15 °C did not
enhance the yield of soluble fusion protein. To enrich
the fusion protein in the soluble fraction, a 65 °C heat-
ing step was performed (Fig. 2, lane 3). This step
allowed most of the unwanted proteins to be irrevers-
ibly precipitated because 500 mg of soluble proteins
were present in the heated supernatant; 70% of which
comprised TRX-huhepc (350 mg). Even though 50%
of the fusion protein was lost in this process, this step,
which takes advantage of the heat resistance of TRX
[33], was found to be necessary to obtain pure TRX-
huhepc.
The TRX-huhepc enriched solution was purified to
homogeneity using affinity TALONÔ Co
2+
chroma-
tography, with the remaining unwanted proteins
flowing through the column (Fig. 2, lane 4) and
TRX-huhepc being eluted with 150 mm imidazole
followed by 2(N-morpholino)ethanesulfonic acid
(MES) buffer (pH 5) (Fig. 2, lane 5), and found to be
Table 1. Primers used for hepcidin cloning and PCR, and primary structure of the purified peptides and proteins. Forward and reverse
oligonucleotides encoding hepcidin sequence contained 5¢ and 3¢ additional nucleotides (in bold) corresponding to the LIC sequence of
pET-32Ek ⁄ LIC vector.
Sequence (5¢-to3¢)
Primers

Mouse hepcidin forward GACGACGACAAGATGGACACCAACTTCCCCATCTGCATCTTCTGCTG
TAAATGCTGTAACAATTCCCAGTGTGGTATCTGTTGCAAAACATAA
Mouse hepcidin reverse GAGGAGAAGCCCGGTTATGTTTTGCAACAGATACCACACTGGGAATT
GTTACAGCATTTACAGCAGAAGATGCAGATGGGGAAGTTGGTGTCCA
Human hepcidin forward GACGACGACAAGATGGACACCCACTTCCCGATCTGCATTTTCTGCTG
CGGCTGCTGTCATCGATCAAAGTGTGGGATGTGCTGCAAGACGTAA
Human hepcidin reverse GAGGAGAAGCCCGGTTACGTCTTGCAGCACATCCCACACTTTGATCG
ATGACAGCAGCCGCAGCAGAAAATGCAGATCGGGAAGTGGGTGTCCA
Proteins
m1hepc MDTNFPICIFCCKCCNNSQCGICCKT
huhepc MDTHFPICIFCCGCCHRSKCGMCCKT
S-m1hepc GSGMETAAKPERQHMDSPDLGTDDDDKMDTNFPICIFCCKCCNNSQCGICCKT
S-huhepc GSGMETAAKPERQHMDSPDLGTDDDDKMDTHFPICIFCCGCCHRSKCGMCCKT
B. Gagliardo et al. Active recombinant hepcidin
FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS 3795
at least 90% pure as measured on the ExperionÔ
device. Routinely, 3.5 mg of fusion protein was
obtained from 1 g of cell pellet.
Step 2: thrombin cleavage
After elution, TRX-huhepc was digested for 16 h with
1 U of thrombin per 10 mg of protein, yielding an
almost complete cleavage (Fig. 2, lane 6). S-huhepc
generated by thrombin digestion was separated away
from TRX using an HPLC purification step on a C18
semi-preparative column. The purified S-huhepc
(Fig. 2, lane 7) was lyophilized to dryness, resuspended
at 1 mgÆmL
)1
in enterokinase cleavage buffer and
treated for 6 h with 10 U of enterokinase per mg of

S-huhepc. Low temperature was avoided to minimize
peptide precipitation during cleavage. Finally, purifica-
tion of huhepc was achieved by anion exchange chro-
matography. As previously noticed for S-huhepc,
huhepc had a propensity to aggregate but remained
soluble in the presence of salts. Thus, the enterokinase
cleaved mixture was simply diluted in one volume of
water to adjust the NaCl concentration to below
40 mm. Under these conditions, low salt buffer at
pH 7, huhepc flowed through the UnoQ anion
exchange column (Fig. 2, lane 8), whereas the other
peptides (S and eventually S-huhepc) bound to the
column. Again, to avoid irreversible aggregation of
huhepc, the huhepc solution was directly lyophilized in
the presence of buffer to maintain further solubility of
the peptide. The peptide was stored as a dry lyophilized
powder until used. When required, remaining salts were
withdrawn either upon thorough dialysis (using
1000 Da molecular weight cut-off dialysis tubing) or
HPLC chromatography (Fig. 3) or size exclusion
chromatography using bio-gelÔ P4 resin (Bio-Rad,
Marnes-la-Coquette, France). Routinely, 2 mg of pure
huhepc was obtained from 60 g of Origami cells.
Mouse hepcidin purification
Mouse hepcidin (m1hepc) was obtained as described
for huhepc, except for minor modifications related to
differences in the pI of the peptide (Fig. 1). Entero-
kinase digestion was carried out at pH 6.2 (instead of
pH 7 for m1hepc) and the UnoQ chromatography step
was run at pH 6.2 rather than pH 7. The production

yield of m1hepc was slightly lower than that of huhepc
(8% instead of 13%, respectively, starting form
Fig. 3. HPLC profile of pure recombinant (A) mouse or (B) human
hepcidins. (A) Fifty microlitres of huhepc (0.1 mgÆmL
)1
) was
injected on a MOS C8 and (B) 50 lL of m1hepc (0.01 mgÆmL
)1
)
was injected on a Vydac C18 column using a stepwise acetonitrile
gradient.
Fig. 2. Purification of huhepc followed by SDS ⁄ PAGE using 12%
Bis-Tris CriterionÔ XT gels in the absence of reducing agent. Large
(left) and small (right) molecular weight markers (Bio-Rad) are indi-
cated in kDa. Lane 1, 200 lg of crude extract of TRX-huhepc
expressing cells; lane 2, 150 lg of TRX-huhepc expressing cells
cytosolic fraction (prior heating step); lane 3, 50 lg cleared cyto-
solic fraction after removal of the heat denaturated proteins; lane 4,
30 lgofCo
2+
TalonÔ unbound protein fraction; lane 5, 50 lgof
Co
2+
TalonÔ 150 mM imidazole eluted fraction, containing TRX-hu-
hepc; lane 6, 40 lg of TRX-huhepc thrombin cleavage mixture;
lane 7, 6 lg of HPLC purified S-huhepc; lane 8, 3 lg of UnoQ
purified huhepc (lyophilized in the presence of buffer). *TRX-huhepc
dimer formed upon disulfide bridge between two thioredoxin
subunits.
Active recombinant hepcidin B. Gagliardo et al.

3796 FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS
TRX-hepc), most likely due to the higher propensity
of m1hepc to precipitate during the enterokinase
cleavage step.
Characterization of the recombinant proteins
and peptides
Purity of the recombinant proteins was assessed by
SDS ⁄ PAGE or ExperionÔ capillary electrophoresis,
HPLC chromatography and MS. As shown in Fig. 2,
the peptides and proteins appeared fairly clean by
SDS ⁄ PAGE analysis (in the absence of reducing agent)
and, importantly, ran at the expected molecular
weights: TRX-huhepc at 20 kDa (Fig. 2, lane 5); S-hu-
hepc at 6 kDa (Fig. 2, lane 7); and huhepc appears to
run as a 3.5 kDa peptide (Fig. 2, lane 8). The extra
sharp band marked with an asterisk, at approximately
37 kDa, corresponded to TRX-huhepc dimers due to
disulfide bridges between two TRX domains. This band
was indeed absent when the SDS ⁄ PAGE was run in the
presence of reducing agents (data not shown). Purified
hepcidin remained soluble and largely as a monomer
after the anion exchange chromatography or when
resuspended in the presence of buffer after lyophiliza-
tion (Fig. 2, lane 8). C18 HPLC analysis also displayed
a single peak for each peptide (Fig. 3, m1hepc and
huhepc), when assessing the purity of the samples.
Interestingly, desalted hepcidins, either obtained
after HPLC purification or desalted after anion
exchange chromatography following dialysis or exclu-
sion chromatography, always aggregated over time.

This phenomenon was observed with low salt solu-
tions, regardless of whether the hepcidins were stored
at 4 °Corat)80 °C and in the presence or absence of
glycerol or detergents. These polymeric hepcidin forms
could not be reverted to the monomer forms, even in
the presence of reducing agents such as dithiothreitol
or tris(2-carboxyethyl)phosphine (data not shown).
Therefore, pure hepcidins were routinely lyophilized
immediately after the anion exchange chromatography
and kept as a salt containing powder. To quantify the
hepcidin content in the lyophilized powder, a sample
was injected on a Vydac (Grace, Templemars, France)
C18 HPLC column, lyophilized again and weighed.
Concentration was also confirmed using UV-visible
measurements [2].
To verify that all the cysteines were engaged in disul-
fide bridges, Ellman’s reagent was used to measure the
content of free cysteines [34]. As indicated in Table 2,
S-m1hepc and S-huhepc, m1hepc and huhepc contained
less than one free cysteine per monomer, indicating that
at least 90% of the hepcidins and hepcidin derivatives
contained the expected four-disulfide bridges.
Finally, the presence of the four-disulfide bridges
was also confirmed using ESI MS (Table 2) for
S-m1hepc and S-huhepc. Upon deconvulation, the
molecular weight was 6020.6 ±1 Da for S-m1hepc and
6057.4 ± 1 Da for S-huhepc, with both values being
in good agreement with the theoretical mass of
6020.82 Da and 6055.94 Da expected for the corre-
sponding S-hepcidins containing four-disulfide bridges.

Unfortunately, ESI MS on pure hepcidins was impos-
sible due to the difficulty of ionizing the peptide.
Therefore, surface enhanced laser desorption ionization
time of flight (SELDI-TOF) MS, previously described
as a powerful technique to analyze hepcidin, was used
[35]. Clean mass spectra were observed when peptides
were run on regular (NP20) protein chips (Table 2).
The calculated mass was 2885.2 ± 1 Da for m1hepc
and 2920.3 ± 1 Da for huhepc, with both values being
in good agreement with the theoretical values of
2885.49 Da and 2920.60 Da, respectively, expected for
fully oxidized disulfide bridged forms, demonstrating
that the recombinant hepcidins were pure and con-
tained four-disulfide bridges. Interestingly, SELDI-
TOF MS performed on aggregated hepcidins (Fig. 4B)
gave values identical to those of the non-aggregated
form, indicating that the polymeric bands observed by
SDS ⁄ PAGE analysis were not related to interchain
disulfide bridges but simple aggregation.
Table 2. Characterization of purified hepcidin derivatives. The calculated average molecular weight was obtained either using ESI MS or
SELDI-TOF MS, as described in the Experimental procedures. ND, not determined; ) , no activity detected or no m ⁄ z signal obtained; +,
activity measured: at least 10 mm diameter growth inhibition of E. coli in plate assay using 100 pmol of each peptide, complete disappear-
ance of ferroportin signal at the membrane of iron treated macrophage J774 cells in the presence of 700 l
M of each of the hepcidin deriva-
tives. Free SH content is related to the lack of detection of free thiols using Ellman’s reagent. All data were obtained for two independent
preparations of hepcidin (MS and SH contents) and three independent preparations for activity measurements.
Name MW (ESI) MW (SELDI-TOF) Free SH (%) Antimicrobial activity Ferroportin activity
S-m1hepc 6020.6 ± 1 6018.9 ± 0.5 < 5 + )
S-huhepc 6057.4 ± 1 ND < 5 + )
m1hepc – 2885.2 ± 0.5 < 3 + +

huhepc – 2920.3 ± 0.5 < 3 + ++
B. Gagliardo et al. Active recombinant hepcidin
FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS 3797
Compared activities of hepcidins and S-hepcidins
The antibacterial activity of m1hepc, huhepc and their
S-tag forms (S-m1hepc and S-huhepc) was tested
against E. coli using a plate assay [27]. In our condi-
tions, 10 lLofa10lm solution for either of the four
peptides routinely resulted in a 10 mm diameter
growth inhibition of E. coli whereas 10 lL of buffer or
medium resulted in no more than a 3 mm diameter
growth inhibition (Table 2). These results demonstrate
that recombinant hepcidins displayed obvious antibac-
terial activity against E. coli and that similar activity
was observed for the S-hepcidins compared to that of
the corresponding hepcidins.
The iron-related bioactivity of the recombinant pro-
teins was tested in Fe-NTA treated J774 macrophages
for their potential to degrade the hepcidin receptor,
ferroportin. As shown by western blotting (Fig. 5),
only iron treated macrophage cells express detectable
amount of membrane bound ferroportin (Fig. 5, lane 1
versus lane 2). As a control, the human synthetic 25
amino acid mature peptide (Peptides International,
Louisville, KY, USA) was used at 700 nm for 5 h. As
expected, a complete disappearance of ferroportin was
observed (Fig. 5, lane 3). When tested under the same
conditions, the huhepc also provoked a complete dis-
appearance of ferroportin (Fig. 5, lane 8). By contrast,
the S-hepcidins were completely inactive towards ferro-

portin degradation (Fig. 5, lanes 4 and 5). These
results were obtained routinely when ‘monomer’ forms
of hepcidin were used in the assay. Interestingly, when
salt-free hepcidins were used (Fig. 5, lanes 6 and 7), no
effect was observed on ferroportin degradation, sug-
gesting that the salt-free hepcidin forms have lost their
biological activity.
The inactivation of hepcidin activity in salt free solu-
tion was further investigated. A fraction of active
UnoQ purified human hepcidin was desalted using a
P4 exclusion chromatography, concentrated, and com-
pared with its parent ‘salted’ hepcidin. The ‘salt-free’
hepcidin form was found completely inactive towards
Fig. 4. (A) Analysis by SDS ⁄ PAGE of UnoQ
purified huhepc before or after ‘desalting’
using a P4 exclusion column settled in
water and concentrated by lyophilization. (B)
SELDI-TOF spectrum of desalted hepcidin.
This result is representative of three differ-
ent experiments performed with external or
internal calibration using low mass peptide
standards (Sigma, St Louis, MO, USA).
Fig. 5. Effects of hepcidin treatment on ferroportin expression in
J774 cells. Expression of ferroportin was studied in macrophage
J774 cells treated with Fe-NTA for 16 h and with the indicated hep-
cidin or S-tagged hepcidin for 5 h. Membrane proteins (30 lg per
lane) were separated by SDS ⁄ PAGE, electro-transferred onto nitro-
cellulose and analyzed with anti-ferroportin serum. The ponceau red
staining of the membrane is shown. Lane 1 is the control without
Fe-NTA and lanes 2 to 8 correspond to samples treated with

Fe-NTA. Lane 3, 700 n
M huhepc from Peptides International; lanes
4 and 5, 100 and 700 n
M of enterokinase digested mixture of
S-m1hepc; lanes 6 and 7, 100 and 700 n
M of pure ‘desalted’
huhepc; lane 8, UnoQ purified salt-containing huhepc (700 n
M).
Active recombinant hepcidin B. Gagliardo et al.
3798 FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS
ferroportin. Interestingly, the migration profile by
SDS ⁄ PAGE of this inactive form of hepcidin showed
an important proportion of multimers (Fig. 4A). Fur-
thermore, the polymerization pattern was identical in
the absence or presence of a reducing agent (not
shown). Altogether, these results clearly demonstrate
that human hepcidin aggregates in the absence of salt,
that this aggregation is not related to intermolecular
disulfide bridge formation (Fig. 4B) and, more impor-
tantly, that human hepcidin activity is significantly
reduced upon aggregation.
Recombinant m1hepc was also shown to degrade
ferroportin, although less efficiently than huhepc. At
100 nm, although both huhepc and synthetic hepcidin
(Peptides International) induced a complete disappear-
ance of ferroportin from the macrophage membranes,
m1hepc yielded only a 50% decrease of ferroportin
under the same conditions. Finally, aggregation-
induced loss of hepcidin activity was also observed
with the m1hepc, to a larger extent than the human

form, most likely due to its slightly more hydrophobic
nature (human hepcidin contains two histidine and one
arginine residues instead of three asparagine residues
for the mouse form).
Discussion
The TRX fusion protein approach in association with
the use of the Origami strain was found to be ideal
for the expression of hepcidins. The recombinant hep-
cidin peptides, containing eight cysteines engaged in
four intramolecular disulfide bridges, were properly
folded, fully oxidized and biologically active. Further-
more, the need of denaturation–renaturation or
reduction–oxidation steps (often present in other pro-
tocols of hepcidin preparation, either recombinant
[26–28] or synthetic [10,17,23]) were removed. By con-
trast to another report [26], our recombinant products
were apparently metal-free. The human and mouse1
hepcidins were purified by two cleavage steps:
removal of TRX with thrombin and S-tagging with
enterokinase. The recombinant products differed from
the native hepcidins by the presence of an extra
amino acid (methionine) at the N-terminus. However,
this extra amino acid did not appear to affect the
biological activities of the recombinant products
because their activities were found comparable to that
of a commercially available synthetic 25 amino acid
human hepcidin (Peptides International). This is in
agreement with observations of Nemeth et al. [17],
who showed that a human hepcidin of 26 amino
acids, bearing an extra alanine in the N-terminal posi-

tion, retained its activity.
Interestingly, S-hepcidins, which bear 29 extra
amino acids upstream of the hepcidin sequence, con-
serve an antibacterial activity comparable to that of
the corresponding hepcidin but are completely ineffi-
cient in degrading the iron exporter ferroportin. This
result is in accordance with observations of Nemeth
et al. [17], who demonstrated that the N-terminal part
of hepcidin was necessary for interaction with ferro-
portin, and strongly suggests that the presence of the
S-tag prevents the hepcidin-ferroportin interaction. It
is thus predictable that any N-terminal tagged recom-
binant hepcidin products, such as GST [26], His [28]
or other fusions, could interfere with hepcidin activ-
ity. Very recently, Koliaraki et al. [29] described the
production of a C-terminal his-tagged human hepci-
din using Pichia pastoris as a host for expression.
Although it was not possible to express untagged
hepcidin, the C-terminal his-tagged recombinant pep-
tide that they produced was able to bind ferroportin
and promote its degradation in Raw 264.7 macro-
phages [29].
The results of the present study also emphasize that
the determination of hepcidin antimicrobial properties
is not relevant to assessing hepcidin bioactivity in iron
metabolism and that only a measurement of hepcidin
activity towards ferroportin degradation and ⁄ of
cellular iron retention should be considered.
In the present study, we have demonstrated that
aggregated forms of hepcidins (dimers or tetramers),

harboring the same SELDI-TOF mass spectrum as
their corresponding monomers, are inactive against
ferroportin. As previously demonstrated [36], hepci-
din derivatives, including the recently described
C-terminal his-tagged peptide [29], are very likely to
precipitate, yet the aggregation does not involve
interchain disulfide bridge formation. This aggregation
occurs at neutral pH in the absence of salt or upon
storage at 4 °C in solution. This phenomenon is
likely to explain the poor reproducibility in hepcidin
preparation (including commercial preparations). In
our hands, the storage of hepcidins as salted lyophi-
lized aliquots at )20 °C (or less) was found to com-
prise the best method for preventing loss of activity
over time. Reproducible preparation of biologically
active hepcidins, either synthetic or recombinant, is a
necessary step for investigating the cellular biology
of this hormone and the present study has contrib-
uted to this aspect. Finally, the strategy described in
the present study was also found to be appropriate
for the production of prohepcidin (B. Gagliardo,
personal communication) and may be useful in
the preparation of other cysteine rich peptides or
proteins.
B. Gagliardo et al. Active recombinant hepcidin
FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS 3799
Experimental procedures
Bacterial strains, media and chemicals
The TG1 E. coli strain [D(lac pro) supE thi hsdD5 F’
traD35 proAB LacIq LacZDM15] was used for cloning and

plasmid DNA purification. Origami B(DE3) cells (Novagen,
Merck Chemicals Ltd, Nottingham, UK) [F
)
ompT hsdS
B
(r
B
)
m
B
)
) gal dcm lacY1 aphC (DE3) gor522::Tn10 trxB
(Kan
R
, Tet
R
)] were used for protein expression.
LB [Bacto-tryptone 1% (w ⁄ v), yeast extract 0.5% (w ⁄ v),
NaCl 5% (w ⁄ v)] and ZYM-5052 [Bacto-tryptone 1% (w ⁄ v),
yeast extract 0.5% (w ⁄ v), 25 mm Na
2
HPO
4
,25mm
KH
2
PO
4
,50mm NH
4

Cl, 5 mm Na
2
SO
4
supplemented with
2mm MgSO
4
, 0.5% glycerol, 0.05% glucose and metal
salts] (1000 · metal salts solution: 50 mm FeCl
3
,20mm
CaCl
2
,10mm MnCl
2
,10mm ZnSO
4
,2mm CoCl
2
,2mm
CuCl
2
,2mm NiCl
2
,2mm Na
2
MoO
4
,2mm Na
2

SeO
3
,
2mm H
3
BO
3
,60mm HCl) were used as regular and high
density culture media, respectively, in accordance with
Studier et al. [32].
Constructions
Human and mouse synthetic oligonucleotides (Table 1) cor-
responding to the hepcidin sequence fused to a LIC site
were synthesized by Eurogentec (Seraing, Belgium).
Mice, unlike humans, have two duplicated genes, Hepc1
and Hepc2 [5,37]. Because the mouse Hepc2 gene was found
unrelated to iron metabolism [37,38], Hepc1 was chosen for
the present study.
Each primer (10 lm) was annealed to its complementary
partner and heated at 90 °C. Temperature was allowed to
decrease to 25 °Cat1°CÆmin
)1
(using a MJ research ther-
mocycler; Bio-Rad). One microlitre of the double stranded
hepcidin DNA was mixed with 1 lL of pET-32Ek⁄ LIC
(50 ng) vector (Novagen) at 25 °C for 5 min and one-tenth
of the mixture was used to electroporate E. coli TG1cells.
Upon transformation, colonies were used to prepare plas-
mid DNA and the presence of pET-32Ek ⁄ LIC-Hepcidin
vector (either mouse or human) was confirmed by

sequencing performed at Genome Express (Meylan,
France).
Expression
The pET-32Ek ⁄ LIC-mouse1hepcidin vector (pET-32LIC-
m1hepc) and the pET-32Ek ⁄ LIC-human hepcidin vector
(pET-32LIC-huhepc) were used to transform ORIGAMI
B(DE3) competent cells. The transformed cells were
cultured in 50 mL of LB medium supplemented with kana-
mycin (10 mgÆL
)1
) and ampicillin (50 mg ÆL
)1
). The precul-
ture was used to inoculate 3.6 L of high-density culture
medium ZYM-5052 [32] supplemented with kanamycin and
ampicillin. The culture was carried out in twelve 2 L Erlen
Flasks at 37 °C for 3 h, then 20–23 h at 25 °C. Isopropyl
thio-b-d-galactoside (1 mm) was finally added and culture
allowed to continue for an extra 3 h at 25 °C. The cells
were harvested at 6000 g for 20 min at 4 °C. Routinely,
the biomass obtained represented 17 gÆL
)1
of wet cells
culture.
Fusion protein purification
TRX-hepcidin containing cells were resuspended in 5 mL
of phosphate buffer (50 mm Na
2
HPO
4

⁄ NaH
2
PO
4
)atpH7
supplemented with NaCl at 300 mmÆg
)1
of cell pellet. Bacte-
ria were lysed in the presence of BugBusterÔ (0.5·) lyso-
zyme (0.5 gÆL
)1
) and DNAse (0.05 mgÆg
)1
of cell pellet),
then sonicated on ice in the presence of phen-
ylmethanesulfonyl fluoride (0.5 mm) and the protease inhib-
itors leupeptin, pepstatin and aprotinin (10 lgÆ mL
)1
each)
using a LabsonicÔ (B. Braun, Melsungen AG, Melsungen,
Germany) device operated at 200 W and with a 0.7 s pulse.
Cells debris were centrifuged at 10 000 g for 20 min. The
resulting supernatant (15–20 mgÆmL
)1
of protein) was
heated to 65 °C and immediately transferred on ice. The
heat inactivated precipitated proteins were removed by
centrifugation at 12 000 g for 15 min and the cleared super-
natant, routinely containing 5 mgÆmL
)1

of protein, was
loaded onto a TALONÔ Co
2+
(Clontech, Ozyme,
St Quentin-en-Yvelines, France) affinity column (15 mL
bed volume). The column was washed with five volumes of
phosphate buffer and the TRX-Hepcidin fusion protein was
eluted with the same buffer completed with imidazole
150 mm (one volume) and 20 mm MES buffer at pH 5 (one
volume).
Cleavage and purification of hepcidin
Affinity purified TRX-hepcidin containing fractions were
pooled and thoroughly dialyzed at 4 °C against 20 mm Tris
buffer (pH 7.4 at 25 °C) supplemented with 500 mm NaCl,
then 1 unit of biotynilated thrombin (Novagen) was added
for 10 mg of fusion protein and cleavage was allowed to
proceed for 16 h at room temperature at a protein concen-
tration of 1.5–3 mgÆmL
)1
. The biotynilated thrombin was
extracted using sreptavidin-agarose and the resulting solu-
tion containing TRX, S-hepcidin and, eventually, some
uncleaved TRX-hepcidin was concentrated to 8 mgÆmL
)1
.
Fractions (1.5 mL) of the concentrated protein solution
were injected onto a BioBasic C18 semi preparative HPLC
column (250 mm · 10 mm, 300 A
˚
; Thermo, Courtabeuf,

France) under the control of a Spectra physics HPLC sys-
tem. The mobile phase run at 3 mLÆmin
)1
consisted of the
water ⁄ acetonitrile gradient: 100% H
2
O [trifluroacetic acid
(TFA) 0.1%] for 10 min, 0–40% CH
3
CN (TFA 0.1%) for
20 min, 40–60% CH
3
CN (TFA 0.1%) for 20 min and 60–
100% CH
3
CN (TFA 0.1%) for 20 min. The S-tag-hepcidin
containing fractions were eluted around 60% H
2
O (TFA
Active recombinant hepcidin B. Gagliardo et al.
3800 FEBS Journal 275 (2008) 3793–3803 ª 2008 The Authors Journal compilation ª 2008 FEBS
0.1%) and 40% CH
3
CN (TFA 0.1%) and lyophilized using
an Alpha 1-LU Lyophilizer (Avantec, Illkirch, France).
S-hepcidin cleavage
S-hepcidin was solubilized at 1 mgÆmL
)1
of protein in
20 mm Tris buffer (pH 6.7) containing 75 mm NaCl, 2 mm

CaCl
2
and 0.05% Chaps and digested for 6 h at room tem-
perature with 10 units of Enterokinase (Novagen) per mg
of S-hepcidin. Enterokinase was removed using the manu-
facturer capture beads and the enterokinase free digestion
mixture was diluted in one volume of H
2
O to decrease the
ionic strength prior to injection onto a UnoQ column (Bio-
Rad) anion exchange column under the control of a Bio-
logicÔ (Bio-Rad) device equipped with a 258 nm detector.
A salt gradient was performed between 20 mm Tris buffer
at pH 7 and 20 mm Tris buffer at pH 7 containing 1 m
NaCl for human hepcidin and at pH 6.2 for the mouse
form. The cleaved recombinant hepcidin, either mouse or
human, was eluted in the void volume. The pure hepcidins
were further characterized by HPLC on a Vydac C18
(250 mm · 4.6 mm, 300 A
˚
pores; Grace, Templemars,
France) reverse phase column. Fifty microlitres of hepcidin
(0.1 mgÆmL
)1
) were eluted with the following gradient at
1mLÆ min
)1
from 90% of (H
2
O, 0.1% TFA) and 10% of

(H
2
O 30%, CH
3
CN 70%, TFA 0.1%) to 10% of (H
2
O,
0.1% TFA) and 90% of (H
2
O 30%, CH
3
CN 70%, TFA
0.1%) for 30 min. M1hepc was eluted at 18 min 20 s and
huhepc at 18 min 45 s.
Protein characterization
Protein concentration was determined using Bradford (Bio-
Rad) reagent [39]; for peptide concentration determination,
Bacitracin (1423 Da) was used as a standard rather than
serum albumin. Hepcidin concentration was determined
either by weighing a salt-free lyophilized powder or using
absorbance spectroscopy (A
214
to A
225
), as described previ-
ously [2]. Purification was followed using SDS ⁄ PAGE anal-
ysis of peptides and small proteins (< 10 kDa) were run on
Criterion 12% BISTRIS XT gels (Bio-Rad) at 200 V and
190 mA for 45 min in MES buffer at pH 7 in the presence
or absence of the reducing agent tris(2-carboxyethyl)phos-

phine. Immediately after migration, gels were treated with
glutaraldehyde (5%, v ⁄ v final) for 20 min and stained using
colloidal Blue [40]. Proteins (> 10 kDa) were characterized
using Capillary Electrophoresis ExperionÔ PRO260 chips
run on an Experion Ô automated electrophoresis station
(Bio-Rad). Free thiol determination was performed follow-
ing the Ellman methodology [34]. The titration curve was
performed using glutathione (4–200 lm) as a standard and
measuring absorbance (A
380
to A
480
) on a Uvikon 420
(Kontron, Zurich, Switzerland) spectrophotometer in the
presence of 5 mm 5,5¢-dithiobis(2-nitrobenzoic acid). Free
thiol concentration was measured on hepcidin samples
using at least 20 lm solutions. Mass spectrometry was run
on a LCQ advantage ion trap (Thermo Finnigan, Courta-
beuf, France) mass spectrometer under an ESI positive
mode (capillary at 275 °C, capillary voltage 21 V, spray
5 kV). SELDI-TOF MS was performed by Photeomics
(Noisy le Grand, France) on a PBS IIc ProteinChip Reader
(Ciphergen Bioystems Inc., Le Raincy, France) using eight-
spot NP20 ProteinChips (Bio-Rad).
Cell culture and western blot analysis
The mouse monocyte-macrophage cell line J774 was cul-
tured as described previously [20]. To increase ferroportin
distribution to the cell membrane prior to hepcidin treat-
ment, cells were incubated for 16 h with Fe-nitrilotriacetate
solution (Fe-NTA; FeCl

3
100 lm-NTA 400 lm). Synthetic
human hepcidin (Peptides International), m1hepc and
huhepc were used at 700 nm for 5 h as described. Protein
extraction and ferroportin detection were performed as
previously described [20].
Acknowledgements
We would to thank Dr Dan Qing Lou for the murine
cDNA prohepcidin construct and Nicole Kubat for
critically reading the manuscript. This study was sup-
ported by funding from ANR (RO06024KK project)
and EEC Framework 6 (LSHM-CT-037296 Euro-
Iron1).
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