Báo cáo khoa học: Recombinant expression of an insulin-like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3 pot

Recombinant expression of an insulin-like peptide 3
(INSL3) precursor and its enzymatic conversion to
mature human INSL3
Xiao Luo
1
, Ross A. D. Bathgate
2,3
, Ya-Li Liu
4
, Xiao-Xia Shao
1
, John D. Wade
2,5
and Zhan-Yun Guo
1
1 Institute of Protein Research, Tongji University, Shanghai, China
2 Howard Florey Institute, University of Melbourne, Australia
3 Department of Biochemistry and Molecular Biology, University of Melbourne, Australia
4 East Hospital, Tongji University, Shanghai, China
5 School of Chemistry, University of Melbourne, Australia
Introduction
Insulin-like peptide 3 (INSL3) is a peptide hormone
member of the insulin superfamily that includes nine
other members in humans, including insulin, insulin-
like growth factor-1 and -2, relaxin-1, -2 and -3, and
INSL4, -5 and -6. INSL3 was cloned in the early
1990s from the cDNA library of the Leydig cells of
the testes, and originally named Leydig cell insulin-
like peptide (Ley I-L) [1–3]. Its cDNA encodes a
Keywords
activity; INSL3; recombinant expression;

refolding; single-chain precursor
Correspondence
J. D. Wade, Howard Florey Institute, The
University of Melbourne, Vic 3010, Australia
Fax: +61 3 9348 1707
Tel: +61 3 8344 7285
E-mail: john.wade@florey.edu.au
Z Y. Guo, Institute of Protein Research,
Tongji University, 1239 Siping Road,
Shanghai 200092, China
Fax: +86 21 658 98403
Tel: +86 21 659 88634
E-mail:
(Received 25 May 2009, accepted 16 July
2009)
doi:10.1111/j.1742-4658.2009.07216.x
Insulin-like peptide 3 (INSL3), which is primarily expressed in the Ley-
dig cells of the testes, is a member of the insulin superfamily of peptide
hormones. One of its primary functions is to initiate and mediate des-
cent of the testes of the male fetus via interaction with its G protein-
coupled receptor, RXFP2. Study of the peptide has relied upon chemical
synthesis of the separate A- and B-chains and subsequent chain recombi-
nation. To establish an alternative approach to the preparation of
human INSL3, we designed and recombinantly expressed a single-chain
INSL3 precursor in Escherichia coli cells. The precursor was solubilized
from the inclusion body, purified almost to homogeneity by immobilized
metal-ion affinity chromatography and refolded efficiently in vitro. The
refolded precursor was subsequently converted to mature human INSL3
by sequential endoproteinase Lys-C and carboxypeptidase B treatment.
CD spectroscopic analysis and peptide mapping showed that the refolded

INSL3 possessed an insulin-like fold with the expected disulfide linkages.
Recombinant human INSL3 demonstrated full activity in stimulating
cAMP activity in RXFP2-expressing cells. Interestingly, the activity of
the single-chain precursor was comparable with that of the mature
two-chain INSL3, suggesting that the receptor-binding region within the
mid- to C-terminal of B-chain is maintained in an active conformation
in the precursor. This study not only provides an efficient approach for
mature INSL3 preparation, but also resulted in the acquisition of a use-
ful single-chain template for additional structural and functional studies
of the peptide.
Abbreviations
GSSG, oxidized glutathione; INSL3, insulin-like peptide 3; IPTG, isopropyl thio-b-
D-galactoside.
FEBS Journal 276 (2009) 5203–5211 ª 2009 The Authors Journal compilation ª 2009 FEBS 5203
prepro-insulin-like polypeptide that contains a signal
peptide, a B-chain, a C-peptide and an A-chain. After
removal of the signal peptide and the C-peptide,
prepro-INSL3 is converted to its two-chain mature
form containing three insulin-like disulfide bonds, two
interchain bonds (A11-B10 and A24-B22) and one
intramolecular bond within the A-chain (A10–A15).
In addition to being primarily expressed in the Leydig
cells of the testes, INSL3 is also expressed in the the-
cal cells of the ovaries [4]. Chemically synthesized
INSL3 shows low cross-reactivity with the relaxin
receptor, RXFP1, but has no cross-reactivity with the
insulin receptor [5]. For this reason, INSL3 has also
been named relaxin-like factor. Male mice homo-
zygous for a targeted deletion of the INSL3 locus
exhibit bilateral cryptorchidism caused by the failure

of gubernaculum development, resulting in abnormal
spermatogenesis and infertility, whereas female homo-
zygotes have impaired fertility associated with deregu-
lation of the oestrus cycle [6,7]. Overexpression of
INSL3 in female mice causes the ovaries to descend
into the inguinal region because of an overdeveloped
gubernaculum [8]. Transgenic mice missing an orphan
leucine-rich repeat-containing G protein-coupled
receptor (LGR8, recently reclassified as the relaxin
family peptide receptor 2, RXFP2) also exhibit crypt-
orchidism, suggesting that INSL3 is probably the nat-
ural ligand of LRG8 [9,10]. Further work confirmed
this deduction [11]. Identification of the INSL3 recep-
tor paved the way for the discovery of receptors for
other relaxin family peptides [12–15]. INSL3 also
mediates the action of luteinizing hormone on the
maturation of oocytes in ovaries and suppression of
the male germ cell apoptosis in the testes [16]. Thus,
INSL3 has important potential roles as a regulator of
fertility, and conversely, LGR8 antagonists have
potential roles as novel contraceptives.
To date, the preparation of INSL3 and its analogues
has relied on solid-phase chemical synthesis of the sep-
arate A- and B-chains and subsequent chain recombi-
nation [17]. To establish an alternative source of
INSL3, in this study, we designed a single-chain
INSL3 precursor and successfully expressed it in Esc-
herichia coli cells. After purification and in vitro refold-
ing, the recombinant precursor was enzymatically
converted to mature human INSL3. Recombinant

INSL3 adopts an insulin-like fold with correct disulfide
linkages and full biological activity. The single-chain
precursor also retains high activity, suggesting it is
kept in an active conformation. This study provides
both an efficient approach for INSL3 preparation, and
also a useful single-chain INSL3 template for struc-
tural and functional studies.
Results
Gene construction, expression and purification of
the single-chain INSL3 precursor
To obtain human INSL3 via recombinant expression, a
single-chain INSL3 precursor was designed as shown in
Fig. 1A,B. In this peptide, the B-chain and A-chain
were linked by an eight-residue linker sequence. For
insulin and insulin-like growth factor-1, the C-terminus
of the B-chain and N-terminus of the A-chain can be
linked by an extremely short peptide (0–2 residues) and
the resultant single-chain molecule can refold well [18–
21]. We deduced that an eight-residue linker would be
sufficient for a similar role in INSL3. A 6· His tag to
facilitate purification was fused at the N-terminus of the
B-chain. Two negative charge clusters to balance the
strong positive charges of INSL3 itself were introduced
into the N-terminus and the linker sequence, respec-
tively. The single-chain precursor was converted to the
double-chain mature human INSL3 by endoproteinase
Lys-C and carboxypeptidase B treatment (Fig. 1B). It
was expected that endoproteinase Lys-C would not be
able to cleave at the carboxyl side of B8K (indicated by
a star) because of steric hindrance. The resulting INSL3

possesses an additional alanine residue at the N-termi-
nus of the B-chain compared with previous chemically
synthesized human INSL3 [5,22,23]. This additional ala-
nine residue is numbered B0, in accordance with the
INSL3 numbering system. For interest, Fig. 1C shows
the solution structure of INSL3 and its insulin ⁄ relaxin-
like fold. It is highly dynamic in solution [23].
The encoding DNA fragment of the human INSL3
precursor was constructed from four chemically synthe-
sized oligonucleotide primers (Fig. 1A), and subse-
quently ligated into a pET expression vector that carries
a6· His tag. E. coli biased codons were used to improve
the expression level of the precursor. The INSL3 precur-
sor was expressed in E. coli strain BL21(DE3) star
under isopropyl thio-b-d-thiogalactoside (IPTG) induc-
tion. As shown in Fig. 2A, after induction by IPTG, a
 12 kDa band (indicated by a star) was significantly
increased, as analysed by tricine SDS ⁄ PAGE. Although
its apparent molecular mass on SDS ⁄ PAGE was slightly
higher than the expected value ( 9 kDa), further anal-
ysis confirmed that it was the precursor of INSL3. After
E. coli cells were lysed by sonication, the precursor was
mainly present in the pellet, as analysed by tricine
SDS ⁄ PAGE (Fig. 2B). The precursor in the pellet was
dissolved by 8 m urea and subsequently purified by
immobilized metal-ion affinity chromatography (Ni
2+
column), as shown in Fig. 2C. As analysed by tricine
SDS ⁄ PAGE (Fig. 2D), the precursor was eluted from
Recombinant expression of an INSL3 precursor X. Luo et al.

5204 FEBS Journal 276 (2009) 5203–5211 ª 2009 The Authors Journal compilation ª 2009 FEBS
the Ni
2+
column by 250 mm imidazole and was almost
homogeneous. The eluted fraction was then dialysed
against water to remove urea and salt.
In vitro refolding of the single-chain INSL3
precursor
The dialysed INSL3 precursor was further purified by
C
18
reverse-phase HPLC (Fig. 3A). Surprisingly, it was
highly heterogeneous on the reverse-phase column
although it showed a predominantly single band on
SDS ⁄ PAGE. After the precursor had been treated with
dithiothreitol to reduce the disulfide bonds, a major
peak (indicated by a star) appeared on the reverse-
phase HPLC (Fig. 3B). An aliquot of this fully
S-reduced INSL3 precursor was modified by reacting
with iodoacetic acid to generate six carboxymethyl
moieties and its identity was confirmed by subsequent
MS analysis (data not shown). Thereafter, the fully
S-reduced INSL3 precursor was refolded in vitro using
oxidized glutathione (GSSG) as a disulfide donor. A
new product (indicated by a double star) appeared on
the HPLC (Fig. 3C) and its measured molecular mass
of 9168.0 Da was consistent with the expected value of
the refolded INSL3 precursor (9168.3). The refolded
single-chain precursor could not be modified by iodo-
acetic acid, as analysed by native PAGE (data not

shown), suggesting that the refolded INSL3 precursor
had acquired three disulfide bonds. The in vitro refold-
ing efficiency calculated from the peak areas of the
reduced and folded INSL3 was  80%, suggesting that
the INSL3 precursor refolded efficiently in vitro.
Enzymatic conversion of the single-chain INSL3
precursor into mature INSL3
To convert the INSL3 precursor into the mature two-
chain human INSL3, the refolded precursor was first
A
BC
Fig. 1. (A) Amino acid sequence and nucleotide sequence of the recombinant human INSL3 precursor. The B-chain and A-chain are shown
in red and green, respectively. The N-terminal 6· His tag and the linker between the B-chain and the A-chain are shown in black. Four oligo-
nucleotide primers (P1, P2, P3 and P4) used to construct the gene of INSL3 precursor are underlined and labelled. The restriction enzyme
cleavage sites (NdeI and EcoRI) are also labelled. (B) Cartoon showing the amino acid sequence of the human INSL3 precursor. The cyste-
ines are shown by filled circles. Disulfide bonds are shown as sticks. The expected Lys-C endoproteinase cleavage sites are indicated by
arrows. B8K that cannot be cleaved by Lys-C endoproteinase because of steric hindrance is indicated by a star. The lysine residue removed
by carboxypeptidase B after Lys-C cleavage at the C-terminus of the B-chain is also indicated. (C) Previously reported solution structure [23]
of human INSL3 (PBD code 2H8B).
X. Luo et al. Recombinant expression of an INSL3 precursor
FEBS Journal 276 (2009) 5203–5211 ª 2009 The Authors Journal compilation ª 2009 FEBS 5205
AB
C
D
Fig. 2. Expression and purification of the human INSL3 precursor.
(A) Analysis of the expression of INSL3 precursor by tricine
SDS ⁄ PAGE. Fifty microlitres of culture broth before and after IPTG
induction were centrifuged and the pellet resuspended in 15 lLof
water and then mixed with 5 lL of loading buffer containing 100 m
M

dithiothreitol. After boiling, the sample was loaded onto a 16.5% tri-
cine SDS ⁄ gel. After electrophoresis, the gel was stained by Comas-
sie Brilliant Blue R250. Lane 1, before induction; lane 2, after
induction. (B) Tricine SDS ⁄ PAGE analysis after sonication. Induced
E. coli cells were lysed by sonication. The total cell lysate (lane 1),
the pellet (lane 2) and the supernatant (lane 3) were loaded onto a
16.5% tricine SDS ⁄ gel, respectively. (C) Purification of INSL3 precur-
sor by immobilized metal-ion affinity chromatography. The pellet of
the cell lysate was dissolved in the lysate buffer (20 m
M phosphate
buffer, pH 7.5, 0.5
M NaCl) containing 8 M urea and 1 mM GSSG.
After centrifugation (10 000 g, 10 min), the supernatant was loaded
onto a Ni
2+
column (1 · 4 cm) and eluted by a step-wise increase in
imidazole concentration in the elution buffer (lysate buffer plus 8
M
urea). The peak of the INSL3 precursor was indicated by a star. (D)
Tricine SDS ⁄ PAGE analysis after immobilized metal-ion affinity chro-
matography. Lane 1, before loading; lane 2, flow-through; lane 3,
eluted by 30 m
M imidazole; lane 4, eluted by 250 mM imidazole.
A
B
C
Fig. 3. In vitro refolding of the human INSL3 precursor. (A) Thirty
microlitres of dialysed INSL3 precursor ( 15 lg) were loaded
onto an analytical C
18

reverse-phase HPLC column, and eluted
with an acetonitrile gradient. (B) Thirty microlitres of dialysed
INSL3 precursor ( 15 lg) were treated with dithiothreitol before
loading onto the analytical C
18
reverse-phase HPLC column. (C)
Thirty microlitres of dialysed INSL3 precursor ( 15 lg) were
sequentially treated with dithiothreitol and GSSG before loading
onto a C
18
reverse-phase HPLC column. Details are given in
Materials and methods.
Recombinant expression of an INSL3 precursor X. Luo et al.
5206 FEBS Journal 276 (2009) 5203–5211 ª 2009 The Authors Journal compilation ª 2009 FEBS
treated by Lys-C endoproteinase which can cleave the
peptide bond at the C-terminal side of lysine residues.
The digestion mixture was analysed by reverse-phase
HPLC as shown in Fig. 4A. The measured molecular
mass of the major peak (indicated by a star) was
6490.0 Da, consistent with the theoretical value
(6489.1) of the expected intermediate which carries
an additional lysine residue at the C-terminus of the
B-chain. Because of steric hindrance, and as expected,
Lys-C endoproteinase cannot cleave at the B8K posi-
tion. Subsequently, the intermediate was further trea-
ted with carboxypeptidase B to remove this additional
lysine residue. The digestion mixture was analysed by
reverse-phase HPLC as shown in Fig. 4B. The mea-
sured molecular mass of the major peak (indicated by
a star) was 6363.0 Da, consistent with the theoretical

value (6363.4) of the mature human INSL3.
Peptide mapping of the refolded single-chain
INSL3 precursor
To determine the disposition of the disulfide linkages,
the refolded INSL3 precursor was first digested by
trypsin that can cleave the peptide bond at the C-ter-
minal side of both lysine and arginine residues. The
digestion mixture was analysed by reverse-phase HPLC
as shown in Fig. 5A. The major peak (indicated by a
star) has a molecular mass of 3832.0 Da, consistent
with the theoretical value (3832.3) of the expected
A
B
Fig. 4. Enzymatic conversion of INSL3 precursor to mature human
INSL3. (A) C
18
reverse-phase HPLC of Lys-C digested INSL3 pre-
cursor. (B) C
18
reverse-phase HPLC of INSL3 precursor sequentially
digested by Lys-C and carboxypeptidase B. One microlitre ( 3 lg)
of digestion mixture was loaded onto a C
18
reverse-phase HPLC
column and eluted with an acetonitrile gradient. The major peak
was manually collected, lyophilized and its molecular mass (MS)
was measured by electrospray MS as shown in (A) and (B). Theo-
retical values are shown in parentheses.
A
B

Fig. 5. Peptide mapping of the refolded human INSL3 precursor.
(A) C
18
reverse-phase HPLC of INSL3 precursor digested by trypsin
at 37 °C for 3 h. (B) C
18
reverse-phase HPLC of INSL3 precursor
sequentially digested by trypsin and Glu-C. The trypsin digestion
product was purified by C
18
column, lyophilized and further
digested by Glu-C endoproteinase at 27 °C for 3 h. The major
peaks were manually collected, lyophilized and their molecular
masses (MS) measured by electrospray MS as shown in (A) and
(B). Theoretical values are shown in parentheses.
X. Luo et al. Recombinant expression of an INSL3 precursor
FEBS Journal 276 (2009) 5203–5211 ª 2009 The Authors Journal compilation ª 2009 FEBS 5207
intermediate (containing disulfide cross-linked B7E-
B16R, B21V-B26R and A9Y-A26Y), suggesting that
the A- and B-chains are linked by interchain disulfide
bonds. Trypsin cannot cleave at the B8K position
because of steric hindrance. The trypsin-digested inter-
mediate was further cleaved by endoproteinase Glu-C
which can cleave the peptide bond at the C-terminal
side of both glutamate and aspartate residues
(Fig. 5B). Peak 3 is the un-digested intermediate, the
measured molecular mass of which was 3832.0 Da.
Peak 2 had a molecular mass of 1407.6 Da, consistent
with the theoretical value (1406.7) of the C-terminal
fragment (containing disulfide cross-linked B21V-B26R

and A20L-A26Y), suggesting that the refolded INSL3
contains disulfide A24-B22. Peak 1 had a measured
molecular mass of 2442.3 Da, consistent with the
theoretical value (2440.1) of the N-terminal fragment
(containing disulfide cross-linked B7E-B16R and A9Y-
A19D), suggesting that B10C forms an interchain
disulfide bond with one cysteine at the N-terminus of
the A-chain.
CD spectroscopic study
The secondary structure of the INSL3 precursor before
and after in vitro refolding was analysed by CD spec-
troscopy. As shown in Fig. 6A, refolding significantly
increased the a-helix content (estimated from CD spec-
tra) of the precursor from 6 to 15%. The secondary
structure of the mature INSL3 was similar to that of
insulin (Fig. 6B), but its a-helix content (28%) esti-
mated from CD spectra was lower that that of insulin
(41%) because of its high dynamics in solution, as
reported previously [23]. The calculated a-helix content
of mature INSL3 is consistent with previously pub-
lished values [22], also suggesting that the refolded
INSL3 has correct disulfide linkages.
Functional cAMP assay
The activity of the mature INSL3 and its precursor
was measured using a receptor-activating assay. Chem-
ically synthesized INSL3 was used as the standard. As
shown in Fig. 7, the recombinant mature INSL3 is
fully active: its pEC50 (10.2 ± 0.07, n = 3) is very
similar to that (10.14 ± 0.12, n = 3) of chemically
synthesized INSL3, suggesting that recombinant

INSL3 is folded correctly. Interestingly, the single-
chain precursor also retained high activity: its pEC50
value being 9.88 ± 0.25 (n = 3), suggesting that the
single-chain precursor can be used as a template for
structural and functional studies of INSL3 because it
can be prepared through recombinant expression more
conveniently and, as well, many analogues can also be
prepared using site-directed mutagenesis.
Discussion
In this study, we designed a single-chain INSL3 pre-
cursor for recombinant expression using a similar
approach to that which was successfully employed for
A
B
Fig. 6. CD spectroscopic study. (A) Far-UV spectra of the human
INSL3 precursor before and after in vitro refolding. (B) Far-UV spec-
tra of the mature human INSL3 and porcine insulin.
Fig. 7. cAMP activity of recombinant INSL3 and its precursor com-
pared to synthetic INSL3. The values are expressed as mean ± -
SEM (n = 3) of three assays performed in triplicate.
Recombinant expression of an INSL3 precursor X. Luo et al.
5208 FEBS Journal 276 (2009) 5203–5211 ª 2009 The Authors Journal compilation ª 2009 FEBS
the recombinant expression of insulin [18,19]. The pre-
cursor misfolded and formed in inclusion body in the
host cells, but it could be solubilized and efficiently
refolded in vitro. Refolded INSL3 was shown to pos-
sess the correct insulin-like disulfide linkages and full
activity, thus confirming the efficiency of the approach
for the preparation of INSL3 and its analogues. It is
expected that a similar strategy can also be used for

the preparation of other insulin superfamily peptides.
E. coli cells are easily cultivated and grown and the
entire culture process only takes  10 h. Under the
current conditions, 1–2 mg of refolded and purified
INSL3 precursor could be obtained from 1 L of the
culture broth. It is expected that further optimization
of the culture conditions should lead to a further
improvement in the expression level of the peptide.
The yield of enzymatic production of mature INSL3
from the single-chain precursor was as high as 85%.
Typically, 0.5–0.6 mg of mature INSL3 could be
obtained from 1.0 mg of precursor. Preliminary efforts
to express the INSL3 precursor in baker yeast were
unsuccessful due to a failure to secrete the peptide
from the transformed yeast cells.
The single-chain INSL3 precursor was shown to
possess near-full RXFP2 receptor activity. The short,
eight-residue linking peptide between the C-terminus
of the B-chain and the N-terminus of the A-chain
obviously does not disrupt the active conformation of
INSL3, in particular the major receptor-binding region
(B27W) at the C-terminus of the B-chain [23,24]. Previ-
ous studies have also shown that chemically synthe-
sized INSL3 retains full activity when its B-chain
C-terminus is anchored to the C-terminus of the
A-chain by a suitable length linker [25,26]. In addition,
recombinant single-chain human relaxin-3 containing a
native 45-residue connecting peptide between the
A- and B-chains was also shown to possess significant
receptor binding activity, again highlighting the reten-

tion of an active conformation [27]. The current
biologically active INSL3 precursor can clearly be used
a template for structural and functional studies of
INSL3 because it can be easily prepared through
recombinant expression. Based on this template, many
INSL3 analogues could be quickly prepared through
site-directed mutagenesis.
Materials and methods
Materials
The oligonucleotide primers were synthesized at Invitrogen
(Shanghai, China). Lys-C endoproteinase, trypsin, Glu-C
endoproteinase and carboxypeptidase B were purchased
from Roche (Mannheim, Germany). Agilent reverse-phase
columns (analytical column: Zorbax 300SB-C18, 4.6 ·
250 mm; semi-preparative column: Zorbax 300SB-C18, 9.4 ·
250 mm) were used in the experiments. The peptide was
eluted from the columns with an acetonitrile gradient com-
posed of solvent A and solvent B. Solvent A was 0.1% aque-
ous trifluoroacetic acid and solvent B was acetonitrile
containing 0.1% trifluoroacetic acid. The elution gradient
was as follows: 0 min, 20% solvent B; 3 min, 20% solvent B;
43 min, 60% solvent B; 45 min, 100% solvent B; 49 min,
100% solvent B, 50 min, 20% solvent B. The flow rate for
the analytical column was 0.5 mLÆmin
)1
and that for the
semi-preparative column was 1.0 mLÆmin
)1
. The eluted pep-
tide was detected by UV absorbance at 280 and 230 nm.

Gene construction, expression and purification of
the single-chain INSL3 precursor
Four chemically synthesized oligonucleotide primers were
annealed, elongated by T4 DNA polymerase, cleaved by
restriction enzymes NdeI and EcoRI, and subsequently
ligated into a pET vector pretreated with same restriction
enzymes. The encoding DNA fragment of the INSL3 pre-
cursor was confirmed by DNA sequencing.
The expression construct (pET ⁄ INSL3) was transformed
into E. coli strain BL21(DE3) star. Transformed cells were
cultured in liquid LB medium (with 100 lgÆmL
)1
ampicil-
lin) to A
600
= 1.0 at 37 °C with vigorous shaking
(250 rpm). IPTG stock solution was then added to a final
concentration of 1.0 mm and the cells continuously cultured
at 37 °C for 8 h with gentle shaking (100 rpm).
E. coli cells were harvested by centrifugation (5000 g,
10 min), resuspended in lysate buffer (20 mm phosphate
buffer, pH 7.5, 0.5 m NaCl) and lysed by sonication. After
centrifugation (10 000 g, 15 min), the pellet was resus-
pended in lysate buffer containing 8 m urea and 1 mm
GSSG. After additional centrifugation (10 000 g, 15 min),
the supernatant was loaded onto a Ni
2+
column that was
pre-equilibrated with the washing buffer (lysate buffer plus
8 m urea). The single-chain INSL3 precursor was eluted

from the column by a step-wise increase in the imidazole
concentration in the washing buffer. The eluted INSL3 pre-
cursor fraction was dialysed (cut-off molecular mass 3 kDa)
against distilled water to remove salt and urea.
In vitro refolding of the single-chain INSL3
precursor
To reduce the disulfide bonds of the INSL3 precursor, 1 ⁄ 10
volume of reduction solution (1.0 m Tris ⁄ HCl, 10 mm
EDTA, 100 mm dithiothreitol, pH 8.7) was added into the
above dialysed precursor solution (the concentration of
INSL3 peptide was  0.5 mgÆmL
)1
). The reduction reaction
was carried out at 37 °C for 1 h. Thereafter, an equal
X. Luo et al. Recombinant expression of an INSL3 precursor
FEBS Journal 276 (2009) 5203–5211 ª 2009 The Authors Journal compilation ª 2009 FEBS 5209
volume of refolding solution (0.1 m Tris ⁄ HCl, 1 mm
EDTA, 40 mm GSSG, pH 8.7) was added to the above
reduction mixture to initiate refolding. Refolding was car-
ried out at 16 °C for 1–2 h. The refolding mixture was
loaded onto a C
18
reverse-phase column and eluted by an
acetonitrile gradient as described above. The eluted frac-
tions were manually collected and lyophilized. The molecu-
lar mass of INSL3 precursor was measured by MS.
Enzymatic conversion of the single-chain INSL3
precursor into mature INSL3
The refolded INSL3 precursor was dissolved in 100 mm
NH

4
HCO
3
buffer (pH 8.3) at a final concentration of
 3mgÆmL
)1
. Endoproteinase Lys-C was then added (one
unit enzyme versus  1 mg INSL3 precursor) and digestion
was carried out at 27 °C for 24–48 h. At different reaction
times, an aliquot (3 lg) was removed and analysed by C
18
reverse-phase HPLC. The eluted peaks were individually
collected and their molecular masses were measured by MS.
Thereafter, carboxypeptidase B was added (emzyme ⁄ pep-
tide mass ratio 1 : 30) to remove the additional lysine resi-
due at the C-terminus of B-chain. The reaction was carried
out at 27 °C for 1 h. Mature INSL3 was purified by C
18
reverse-phase HPLC, lyophilized and its molecular mass
determined by MS.
Peptide mapping of the refolded single-chain
INSL3 precursor
The refolded INSL3 precursor was first digested by trypsin
(enzyme ⁄ peptide mass ratio 1 : 10) at 37 °C. At different
reaction times, an aliquot ( 3 lg) was removed and analy-
sed by C
18
reverse-phase HPLC. The eluted peaks were col-
lected separately, lyophilized and their molecular masses
measured by MS. Thereafter, the trypsin-digested product

was further cleaved by Glu-C endoproteinase (enzyme ⁄ pep-
tide mass ratio 1 : 10) at 27 °C. At different reaction times,
an aliquot ( 2 lg) was removed and analysed by C
18
reverse-phase HPLC. The eluted peaks were manually col-
lected and their molecular masses were measured by MS.
CD spectroscopic study
The INSL3 precursor and mature INSL3 were dissolved in
20 mm phosphate buffer (pH 7.4) and their concentration
determined by UV absorbance at 280 nm using an extinc-
tion coefficient of e
280
= 8480 m
)1
Æcm
)1
that is calculated
from the number of tryptophan and tyrosine residues in
INSL3. Their final concentrations were adjusted to 25 lm
for CD measurement which was performed on a Jasco-715
CD spectrometer at room temperature. The spectra were
scanned from 250 to 190 nm with a cell of 0.1 cm path
length. The software j-700 for windows secondary
structural estimation (v. 1.10.00) was used for second-
ary structural content evaluation from CD spectra.
Functional cAMP assay
The cAMP activity assay using HEK-293T cells stably
transfected with human RXFP2 was performed as previ-
ously described [28]. The data were analysed using graph-
pad prism 4 and are the mean ± SEM of three

independent assays performed in triplicate.
Acknowledgments
This work was supported by the Science and Technol-
ogy Commission of Shanghai Municipality (07pj14082)
and the National Natural Science Foundation of
China (30700124). The studies carried out at the How-
ard Florey Institute, Australia, were supported by
NHMRC project grants (#509048 and #454375) to
JDW and RAB.
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