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: 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. 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. References 1 Adham IM, Burkhardt E, Benahmed M & Engel W (1993) Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells. J Biol Chem 268, 26668–26672. 2 Burkhardt E, Adham IM, Hobohm U, Murphy D, Sander C & Engel W (1994) A human cDNA coding for the Leydig insulin-like peptide (Ley I-L). Hum Genet 94, 91–94. 3 Burkhardt E, Adham IM, Brosig B, Gastmann A, Mattei MG & Engel W (1994) Structural organization of the porcine and human genes coding for a Leydig cell-specific insulin-like peptide (LEY I-L) and chromosomal localization of the human gene (INSL3). 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Recombinant expression of an INSL3 precursor FEBS Journal 276 (2009) 5203–5211 ª 2009 The Authors Journal compilation ª 2009 FEBS 5211 . 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 ,. 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. . 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