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

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 refolded pr

(INSL3) precursor and its enzymatic conversion to

mature human INSL3

Xiao Luo1, Ross A D Bathgate2,3, Ya-Li Liu4, Xiao-Xia Shao1, John D Wade2,5and Zhan-Yun Guo1

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: zhan-yun.guo@tongji.edu.cn

(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.

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] Chemithe-cally 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 receprecep-tors 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 colicells 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

a 6· 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 colicells 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 (Ni2+ column), as shown in Fig 2C As analysed by tricine SDS⁄ PAGE (Fig 2D), the precursor was eluted from

the Ni2+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

C18reverse-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

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).

A B

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 lL of

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 m M 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 m M 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 C18 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 C18 reverse-phase HPLC column (C) Thirty microlitres of dialysed INSL3 precursor ( 15 lg) were sequentially treated with dithiothreitol and GSSG before loading onto a C18 reverse-phase HPLC column Details are given in Materials and methods.

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) C18 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 C18 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) C18reverse-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 C18 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.

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 compared to synthetic INSL3 The values are expressed as mean ± -SEM (n = 3) of three assays performed in triplicate.

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 conformareten-tion [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 A600= 1.0 at 37C 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 37C 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 Ni2+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 37C for 1 h Thereafter, an equal

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 16C for 1–2 h The refolding mixture was

loaded onto a C18 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

NH4HCO3 buffer (pH 8.3) at a final concentration of

 3 mgÆmL)1 Endoproteinase Lys-C was then added (one

unit enzyme versus 1 mg INSL3 precursor) and digestion

was carried out at 27C for 24–48 h At different reaction

times, an aliquot (3 lg) was removed and analysed by C18

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 27C for 1 h Mature INSL3 was purified by C18

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 C18reverse-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 27C At different reaction times,

an aliquot ( 2 lg) was removed and analysed by C18

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 e280= 8480 m)1Æcm)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

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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