High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies

Protein Expression and PuriWcation 48 (2006) 253–260 www.elsevier.com/locate/yprep

High yield expression and puriWcation of isotopically labelled human endothelin-1 for use in NMR studies Thien-Thi Mac a, Michael Beyermann b, José Ricardo Pires c, Peter Schmieder a, Hartmut Oschkinat a,¤ a

Abt. NMR-unterstützte Strukturbiologie, Forschungsinstitut für molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany b Abt. Peptidchemie, Forschungsinstitut für molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany c Structural Biology Programme, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Av. Brigadeiro Trompowiski s/n CCS, Rio de Janeiro, RJ 21941-590, Brazil Received 16 December 2005, and in revised form 23 January 2006 Available online 20 February 2006

Abstract Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with signiWcant pharmacological importance. An eYcient and straightforward expression strategy that enables cost-eVective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-eVective expression system in Escherichia coli for the production of ET-1 enriched with 15N and 13C isotopes. Employing thioredoxin as carrier protein, speciWc and nearly quantitative cleavage of ET-1 from the fusion was mediated by Factor Xa, and puriWcation to homogeneity (Wnal purity of >95%) was achieved by RP-HPLC. PuriWed recombinant ET-1 was found to be indistinguishable from the synthetic counterpart as determined by mass spectrometry and NMR spectroscopy. Our expression strategy oVers the potential for production of isotopically labeled ET-1 in large (mg) quantities for the purpose of heteronuclear NMR experiments. Moreover, the method devised should be applicable for recombinant expression of small peptides in general. © 2006 Elsevier Inc. All rights reserved. Keywords: Endothelin-1; DisulWdes; Peptide production; Isotopic labelling

Endothelin hormones and their cognate receptors are involved in many disease states including carcinogenesis, bronchoconstriction, Wbrosis, heart failure, and pulmonary hypertension [1]. Upon interaction of endothelins with receptors ETAR and ETBR, which belong to the superfamily of G-protein coupled receptors, a variety of physiological eVects are elicited such as vasoconstriction, cellular development, diVerentiation, and mitogenesis [2]. Endothelin-1 (ET-1)1 is a peptide consisting of 21 amino acids including four cysteine residues forming two intra*

molecular disulWde bonds between cysteines 1/15 and 3/11. The ET-1 precursor is initially expressed as a 212residue protein, preproET-1, which is processed by successive enzymatic cleavages to the 38-residue intermediate bigET-1 that is Wnally cleaved by endothelin converting enzymes to the mature ET-1 peptide [3]. There has been signiWcant interest in the availability of human ET-1 in high yield, purity, and biological activity. Although the production of peptides, such as ET-1 may be achieved via solid-phase chemical synthesis, this method is

Corresponding author. Fax: +49 30 94793169. E-mail address: [email protected] (H. Oschkinat). 1 Abbreviations used: a.m.u., atomic mass unit; ET-1, endothelin 1; ETAR/ETBR, endothelin receptor subtype A/subtype B; DQF-COSY, double quantum Wltered correlation spectroscopy; HSQC, heteronuclear single quantum coherence; IPTG, isopropylthiogalactoside; MALDI-TOF, matrix-assisted laser desorption/ionization time of Xight mass spectroscopy; m/z, mass to charge NMR, nuclear magnetic resonance; RP-FPLC, reverse-phase fast protein liquid chromatography; RP-HPLC, reverse-phase high performance liquid chromatography; TCEP, Tris(2-carboxyethyl)-phosphine hydrochloride; TOCSY, total correlation spectroscopy; Trx, thioredoxin. 1046-5928/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.01.022

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not suitable to obtain uniformly or partially isotopically (13C, 15N, and 2H) labeled endothelin peptides, which are required to elucidate biophysical properties of the ligand– receptor interaction by NMR spectroscopy [4]. An alternative to chemical synthesis with the opportunity to incorporate isotopic labels is the recombinant expression of peptides. In Escherichia coli (E. coli), expression of peptides or proteins below 7 kDa is known to be hampered by protein instability and degradation attributed to an unstructured state in solution that is susceptible to cellular proteases [5]. Accordingly, direct expression of ET-1 has not been successful, as reported [6]. To circumvent this limitation, the peptide can be expressed as minor part of a fusion protein which aids in overall stability and also solubility. For the recombinant expression and puriWcation of bigET-1, several systems have been developed, which employed fusions of bigET-1 to alkaline phosphatase [7], maltose binding protein [8], or the N-terminal domain of -galactosidase [9–11]. Following expression of the fusion protein, bigET-1 was released from the fusion partner by cleavage using either trypsin (cleavage after Lys or Arg; [8,9,11]), or Factor Xa (cleavage site after IleGlu-Gly-Arg; [10]) or a combination of collagenase (cleavage site after Gly-Pro in (Gly-Pro-Ala)4) and dipeptidylpeptidase IV (removal of Gly-Pro; [7]). Finally, recombinant ET-1 was described to be obtained from bigET-1 by proteolysis with -chymotrypsin [8], or pepsin [9]. As apparent from the multitude of reports, no consensus has been reached as to the optimal expression, puriWcation, and proteolysis strategy. In particular, a method for direct puriWcation of mature ET-1 from a fusion partner (omitting the intermediate step of bigET expression) has been missing so far. In this study, our aim was to establish an eYcient and cost eVective system for (i) expression in E. coli, (ii) puriWcation, and (iii) isotopic labelling of ET-1. To this end, expression constructs encoding fusion proteins of thioredoxin (Trx) to the 21 amino acid ET-1 were analyzed and a (His)6 tag for aYnity puriWcation was incorporated. Almost complete cleavage of the fusion protein was obtained by digestion with Factor Xa. The released ET-1 was puriWed to homogeneity by preparative RP-HPLC yielding a Wnal purity of at least 95%. PuriWed recombinant ET-1 was found to be indistinguishable from the chemically synthesized human ET-1 as determined by mass spectrometry and 2D 1H NMR spectroscopy. Our expression protocol will provide the basic requirements for NMR studies of isotopically labelled ET-1 bound to its cognate receptors, the ETAR or ETBR. Materials and methods Plasmid construction The expression plasmid pET32b-TrxXaET-1 was constructed by replacing the Enterokinase recognition site by a Factor Xa site in the expression vector pET32b-TrxEKET-

1. In a Wrst cloning step, a 93 bp PCR ampliWcation product using the primers 5⬘_NspV_Xa_ET-1 (5⬘- TTCGAAATCG AGGGAAGGTGCTCCTGCTCGTCCCTGATG-3⬘) and 3⬘_HindIII(Stop)ET-1_overhang (5⬘-CCCAAGCTTTCA CCAAATGATGTCCAGGTGGCAG-3⬘) and pET32bTrxEKET-1 as template was cloned into the vector pGEMTeasy (via T/A overhangs; Promega). The 12 bp Factor Xa site was introduced by the 5⬘ primer. In a second cloning step, this intermediate (pGEMTeasy-Xa-ET-1) was cut with BstBI and HindIII and the insert was cloned into the plasmid pET32b-TrxEKET-1 which was previously opened with BstBI and HindIII. The Wnal expression vector pET32b-TrxXaET-1 was sequence-veriWed in both directions using the given primers as sequencing primers. Expression of Trx-Xa-ET-1 fusion proteins Trx-Xa-ET-1 fusion proteins were expressed using an E. coli BL21 (DE3) host (Novagen) and M9 minimal medium. Prior to growth on this medium starter cultures were grown overnight in 5 ml of LB medium containing 100 g/ml ampicillin at 37 °C. Starter cultures were adapted for 24 h in 50 ml of M9 minimal medium containing 0.5 g/L NH4Cl and 3 g/L glucose at 37 °C using 1% inoculum. On the following day, adapted cultures were subcultured using 2£ 500 ml of M9 minimal medium and 1% inoculum. For 15 N enrichment, M9 was supplemented in the Wnal culture with 0.5 g/L [15N]NH4Cl as the sole source of nitrogen. For uniform 13C enrichment, additionally 3 g/L [13C]glucose were provided as the sole source of carbon and for partial 13 C enrichment either 3 g/L 1,3-[13C]glycerol and 2 g/L NaHCO3, or 3 g/L 2-[13C]glycerol and 2 g/L NaH13CO3 were supplemented. After growing the cells to an OD600 of 1.0–1.1, the cultures were induced for recombinant protein expression with isopropylthiogalactoside (IPTG; 1 mM) and the temperature was reduced to 25 °C. Cells were harvested 4 h later, washed once with 0.9% NaCl, pelleted and stored at ¡70 °C. PuriWcation of Trx-Xa-ET-1 fusion proteins All work was carried out at 4 °C. E. coli cells were thawed and resuspended in lysis buVer (20 mM Tris/HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 2 mM -mercaptoethanol) and a protease inhibitor cocktail (“complete”; Roche Diagnostics), lysed in a French pressure cell and centrifuged two times at 75,000g for 20 min to remove all insoluble cellular debris. Supernatant was loaded onto a 50 ml column of Ni–NTA superXow resin (Qiagen). Following pre-equilibration with lysis buVer and loading of the supernatant, the column was washed with three column volumes of the lysis buVer and then with two column volumes of lysis buVer additionally containing 50 mM imidazole. Elution of the fusion protein was carried out with lysis buVer additionally containing 100 mM imidazole. Eluted protein solution was concentrated to a volume of 30 ml (Vivaspin concentrator, 5 kDa molecular mass cutoV, Vivascience). A

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second puriWcation step using 6 ml anion exchanger ResourceQ (Amersham Pharmacia) was carried out. The ResourceQ column was loaded, washed with buVer containing 20 mM Tris/HCl pH 7.0, 1 mM EDTA, and 2 mM -mercaptoethanol and elution of fractions containing puriWed fusion protein was performed with the washing buVer supplemented with 450 mM NaCl. After concentration to a volume of 30 ml (Vivaspin concentrator) the homogeneity of the puriWed protein was conWrmed by SDS–PAGE. Finally, the concentration of Trx-Xa-ET-1 was determined by UV spectroscopy (280 nm;  D 21,273 M¡1 cm¡1). Proteolytic cleavage and puriWcation of ET-1 from the fusion protein For cleavage with Factor Xa the puriWed protein (1– 2 mg/ml) was digested using 1 U of Factor Xa (Qiagen) for 0.1 mg fusion protein at 23 °C for 16–24 h in buVer containing 20 mM Tris/HCl (pH 7.2), 50 mM NaCl, 1 mM CaCl2, and 2 mM -mercaptoethanol. Following proteolytic cleavage, fragments were separated by RP-HPLC (LDC-Analytical, Pickering Laboratories), using a phenomenex Jupiter C18 preparative column (250 mm £ 21.2 mm, 300 Å, 5 m spherical particle size) with a Xow rate of 5 ml/min and UV-detection at 220 nm. The column was equilibrated with 10% acetonitrile in ddH20/0.1% TFA until a stable baseline was attained. Five milliliters of 1– 2 mg/ml protein was subsequently loaded onto the column. The products were eluted with a gradient of acetonitrile from 10 to 95% over 70 min, 6–8 runs were performed, and a total of up to 5–6 mg of puriWed ET-1 was recovered. Fractions collected from the preparative run were analytically assayed using the same column and synthetic ET-1 as external standard. Pure fractions were pooled, lyophilized to a white powder, and stored at ¡20 °C. The concentration of puriWed ET-1 was determined by UV spectroscopy (280 nm;  D 2793 M¡1 cm¡1). Synthetic ET-1 was prepared by Fmoc solid phase synthesis in the group of Dr. M. Beyermann (FMP, Berlin). MALDI-TOF A Voyager-DE STR Biospectrometry workstation with delayed extraction and reXectron capability (PE Biosystems) was used. Mass spectra were obtained in the positive ion mode and operated in reXector mode. Twenty kilovolt accelerating voltage was used. To estimate the eYciency of isotope incorporation, the ratios of the signal intensities of the observed monoisotopic mass and the theoretical monoisotopic mass of complete uniformly labelled ET-1 were calculated. In addition the obtained isotope distribution of the uniformly labelled ET-1 was compared with a predicted distribution using the isotope simulator IsoPro 3.0 (http:// members.aol.com/msmssoft/). For reduction of cysteine residues, the ET-1 sample was incubated with a 30-fold molar excess of tris(2-carboxyethyl)-phosphine hydrochloride (TCEP; Pierce) in acetic acid/H2O (40:60 v/v), pH 2.5, at

255

room temperature for 30 min. The samples were subjected immediately to MALDI-TOF measurements. NMR spectroscopy For all NMR experiments, measurements were performed using a Bruker DRX600 spectrometer at 298 K. Samples (unlabelled, uniform [15N and 13C] or selective 13C labelled ET-1) were dissolved in 0.5 ml acetic acid-d3/H2O (40:60 v/v) at pH 2.5 to give a Wnal peptide concentration of 0.7–1.7 mM. A total correlation spectroscopy (2D-TOCSY) experiment was performed, collecting 1024–2048 points in f2 and 400–600 points in f1. A 15N HSQC spectrum was recorded with 1024 (t1) £ 512 (t2) complex points in each dimension and spectral width of 3012 Hz (15N) £ 10,000 Hz (1H). Backbone C, HN, N and side chain C resonances were assigned, using triple resonance experiments (HNCACB and HN(CO)CACB pair). For all experiments performed, quadrature detection in the indirect dimensions was achieved using time-proportional phase incrementation (TPPI) and solvent suppression was carried out by using the presaturation method or using the Watergate pulse sequence. The spectra were processed with the software package XWINNMR version 2.6 (Bruker), whereas the assignment of the resonances was carried out using Sparky version 3.100 (T.D. Goddard & D.G. Kneller, University of California). Results and discussion Expression and puriWcation of isotopically labelled Trx-Xa-ET-1 fusion proteins To devise a straightforward expression strategy for human ET-1 in E. coli, we generated the construct pET32bTrxXaET-1 (Fig. 1 A) encoding a fusion of ET-1 to the C-terminus of thioredoxin, a (His)6 tag for aYnity puriWcation and a Factor Xa cleavage site between the fusion partners. Thioredoxin was selected as fusion partner because of its small size (109 amino acids), high expression level in bacterial cells [12], and its property to act as a chaperone to protect against undesirable aggregation during expression [13]. In general, conditions for expression and puriWcation were initially optimized in M9 minimal medium and Wnally applied for expression of labelled ET-1 by supplementing M9 medium with 15N and 13C sources (as described in Materials and methods). Upon expression of the construct in the E. coli strain BL21(DE3), the soluble and insoluble cell extracts were separately analyzed by SDS–PAGE. A protein species exhibiting approximately the molecular mass calculated for the His6-TrxXaET-1 (18.1 kDa) was found to be overexpressed and to remain to a large extent in the soluble fraction (Fig. 1B). The soluble fraction was puriWed Wrst by Ni-NTA aYnity chromatography and subsequently by anion exchange chromatography. In the majority of eluate fractions analyzed from the Wnal puriWcation step by

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A pET32bTrxXaET-1 T7 Pr.

Thioredoxin

His6 21

327

Bp:

B

Xa

ET-1

12

63

60 18

C

Fig. 1. Expression and puriWcation of the Trx-Xa-ET-1 fusion protein in E. coli. (A) Schematic structure of the expression construct. Sequences encoding the thioredoxin domain, the Factor Xa recognition site, the (His)6 tag and human ET-1 (represented as boxes; size in bp given beneath) are placed under control of the T7 promoter. (B) SDS–PAGE analysis of the fusion protein synthesized in E. coli. Lanes 1–3 correspond, respectively, to whole cell lysate, soluble fraction of cell lysate and puriWed inclusion bodies. Protein was detected by Coomassie Blue staining. (C) SDS–PAGE analysis of eluted fraction from the Wnal puriWcation of Trx-Xa-ET-1 by anion exchange chromatography. Lanes correspond, respectively, to the Trx-Xa-ET-1 sample loaded (lane 1), Xow-through (lane 2), wash-fraction (lane 3), and eluate fractions (lanes 4–8). Protein was detected by silver staining.

SDS–PAGE (Fig. 1C), a single protein species with an apparent molecular mass of about 18 kDa was detected. A higher resolution of the molecular mass of this protein species was determined by MALDI-TOF which showed a predominant molecular species of 18,100 § 100 Da. An additionally observed protein species of apparently the double size (lane 8 in Fig. 1C) resulted possibly from the formation of dimers covalently linked by cysteine disulWde bonds, since both protein species were recognized by an antibody directed against the (His)6 tag (data from Western Blot analysis not shown). The total concentrations of puriWed Trx-Xa-ET-1 fusion proteins were determined by UV spectroscopy (Table 1). The yield varied between 80 mg/L ([13C]glucose), 61 mg/L (1,3-[13C]glycerol) and 65 mg/L (2[13C]glycerol), respectively, pointing towards a higher metabolic eYciency of glucose compared to glycerol as sole carbon source. Cleavage of the Trx-Xa-ET-1 fusion protein by Factor Xa and RP-HPLC analysis Our goal was to isolate from the fusion protein a cleavage product with a primary sequence identical to authentic ET-1. Therefore we decided to select a proteolysis strategy using Factor Xa that is known to cleave with high speciWcity

N-terminally to the tetrapeptide Ile-Glu-Gly-Arg thereby converting the puriWed precursor to the 21 amino acid ET1. The conversion eYciency of the fusion protein to the cleavage product was approximately 90% as estimated from SDS–PAGE (data not shown). To separate the cleavage products, we employed preparative RP-HPLC and to identify the correct 21 amino acid ET-1 species, we made use of synthetically available ET-1 peptide as an external reference. Separation chromatograms of the isotopically labeled Trx-Xa-ET-1 cleavage products in comparison to synthetic ET-1 show that both samples gave rise to a single and sharp peak elution at an acetonitrile concentration of 24% corresponding to a retention time of 44.5 min. The identity in terms of retention time conWrmed the recovery of a fragment with physical properties (hydrophobicity) identical to synthetic endothelin. Furthermore, this result strongly suggests conformational identity of both species with respect to their three-dimensional structures. The yield of puriWed ET-1 was determined by UV spectroscopy (Table 1). Taking into account the molar weight ratios of fusion protein and released peptide, the total yield of puriWed ET-1 with respect to puriWed Trx-Xa-ET-1 was as high as 57%. This result demonstrates for the Wrst time that isotopically labelled ET-1 can be eYciently produced employing a single cleavage step in mg quantities.

Table 1 Comparison of recombinant ET-1 protein production yield [13C6]Glucose Total protein Fusion protein (Trx-Xa-ET-1) Isolated ET-1

64 mg (3.5 mol) 5 mg (2 mol)

1,3-[13C]Glycerol Yield

Total protein

57%

61 mg (3.3 mol) 3.6 mg (1.4 mol)

2-[13C]Glycerol Yield

Total protein

Yield

42%

65 mg (3.5 mol) 2.0 mg (0.8 mol)

23%

Fusion proteins were isolated from 0.8 L ([13C]glucose) and 1 L ([13C]glycerol) cultures. Total protein (mg), molarity (mol), and yield (%) are given.

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MALDI-TOF analysis of labelled ET-1 An important question for applications using isotopically labelled proteins is to what extent the isotopes were incorporated. To analyze the extent of 15N and 13C incorporation, we subjected the puriWed ET-1 samples to a MALDI-TOF mass spectrometry. Whereas [15N]NH4Cl and [13C]glucose substrates are expected to result in uniform labelling of all N and C atoms, respectively, the [13C] from 1,3-[13C]glycerol, and 2-[13C]glycerol, when these compounds are used as carbon sources, is expected to be partially incorporated into diVerent amino acids [14].

We calculated the mass of ET-1 containing all four cysteine residues in the oxidized state to be at 2491.03 Da. This value is in good accordance to the major peak for recombinant non-labelled ET-1 (m/z D 2490.67; Fig. 2A) considering an error range of 0.5 Da generally assumed for peptide MALDI-TOF measurements in reXector mode. This result conWrms the identity to authentic ET-1 of the RP-HPLCpuriWed cleavage product. An analysis of isotopic incorporation is based on the shift in m/z for the isotopically labeled ET-1. However, also the reduction of the four cysteines would lead to an incremental increase in peptide mass and therefore compromise B

A 100 90 80 70 60 50 40 30 20 10 0 699.0

1359.4

2019.8 2680.2 Mass (m/z)

3340.6

0 4001.0

1.3E+4

2533.8862

1359.4

2019.8 2680.2 Mass (m/z)

3340.6

0 4001.0

2487.2

2490.4 2493.6 Mass (m/z)

1.3E+4

2496.8

0 2500.0

2494.9440

100 90 80 70 60 50 40 30 20 10 0 2490.0

1.3E+4

2493.8

2497.6 2501.4 Mass (m/z)

2505.2

0 2509.0

F

E 100 90 80 70 60 50 40 30 20 10 0 699.0

2624.2048

1876.0185

1359.4

2644.1981

2019.8 2680.2 Mass (m/z)

G

3340.6

2628.3432

0 4001.0

3576.4

1875.0736

1359.4

2624.3676

2019.8 2680.2 Mass (m/z)

3340.6

0 4001.0

100 90 80 70 60 50 40 30 20 10 0 2615

2624.2048

2619

2623 2627 Mass (m/z)

H

% Intensity

100 90 80 70 60 50 40 30 20 10 0 699.0

9.7E+3

% Intensity

% Intensity

100 90 80 70 60 50 40 30 20 10 0 2484.0

D

2494.9440

% Intensity

% Intensity

2528.6423 2512.6327

1781.6659

C 100 90 80 70 60 50 40 30 20 10 0 699.0

2490.6667

1.3E+4 % Intensity

% Intensity

2490.6667

% Intensity

257

100 90 80 70 60 50 40 30 20 10 0 2622.0

9.7E+3

2631

2628.3432

0 2635

3576.4

2624.3676

2624.8

2627.6 2630.4 Mass (m/z)

2633.2

0 2636.0

Fig. 2. MALDI-TOF mass spectrometry analysis of puriWed ET-1. Mass spectrum of recombinant unlabelled ET-1 before (A) and after reduction (C). Associated sodium and potassium adducts may be responsible for side peaks at m/z 2512.63 and 2528.64, respectively. Enlarged view shows the monoisotopic mass proWle of oxidized (B) and reduced (D) unlabelled ET-1. An isotopic multiplet results from unique abundances of naturally occuring C, H, N, O, and S isotopes. This shows the mass shift of 4 Da. Mass spectrum of uniformly 13C/15N labelled recombinant ET-1 before (E) and after reduction (G) with enlargements showing the same species (F) or its reduced derivative (H).

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calculation of isotopic incorporation. To determine the extent of disulWde bond formation in recombinant ET-1 from the change in the m/z ratio upon disulWde reduction, we compared the mass spectra before and after reduction for samples of both the recombinant unlabelled and labelled ET-1. A comparison of the mass spectra obtained for puriWed unlabelled ET-1 and its reduced derivative (enlarged monoisotopic mass proWles shown in Figs. 2B and C) conWrms a diVerence in m/z of 4 a.m.u. whereas the overall distribution of the multiplet seemed to be unaVected. This result indicates that the cysteine residues in recombinant ET-1 are homogenously oxidized (two intrachain disulWde bonds) whereas the cysteine residues in the TCEP-treated sample are homogenously reduced. The MALDI-TOF spectrum for ET-1 that has been uniformely labelled using [15N]NH4Cl and [13C]glucose as substrates shows a predominant peak with a mass (m/z) of 2624.09 Da (Fig. 2D). Assuming complete isotope incorporation, we calculated the monoisotopic mass for this species to be at 2624.31 Da which is in good accordance to the experimentally obtained m/z value. Upon reduction, an increase in 4 a.m.u. was also observed for 13C- and 15Nlabelled ET-1 (compare multiplet proWles in Fig. 2E (oxidized) and Fig. 2F (reduced)) conWrming that the recombinant species contains homogenously oxidized cysteine residues. To determine the percent incorporation of 13 C and 15N isotopes, we applied two diVerent methods: Wrst, we compared the intensity ratios of peaks at incrementally (¡1 a.m.u.) lower masses to the peak at 2624 Da (complete labelling). Secondly, we compared our experimental monoisotopic mass distribution proWle to a predicted proWle using the isotope simulator IsoPro 3.0. Both methods indicated an average 13C- and 15N-labelling of 798%. In an analogous way, we subjected ET-1 samples labelled with 1,3-[13C]glycerol and 2-[13C]glycerol to MALDI-TOF spectroscopy and observed predominant species at 2583.23 and 2558.91 Da, respectively (mass spectra not shown). With respect to 15N incorporation, conditions of bacterial expression were as described before and thus a similar degree of 15N incorporation may be assumed. However, the pattern of 13C-labelling in these ET-1 species is complex and dependent on the anabolism of individual amino acids [14] preventing accurate determination of the degree of 13C incorporation. NMR analysis of labelled ET-1 NMR spectroscopy can provide insight on purity, conformation, and folding states of the puriWed peptide, as additional information over the mass spectrometric results. For synthetic ET-1, the sequence-speciWc 1H NMR assignments as determined by DQF-COSY have already been described [15], and provided a reference to NMR spectra of our samples. From 1H NMR TOCSY experiments we determined the chemical shifts for synthetic and recombinant ET-1 (Fig. 3A). First, we compared the 1H NMR chemical shifts

A synthetic

recombinant

B S5 C15

M7

E10 S2

H16

D18

C3

F14 S4

D8 C11 I 19

L17

Y13 V12

K9

I2 0

L6 W21sc

W21

Fig. 3. NMR analysis of recombinant ET-1. (A) NH Wngerprint regions of the 2D-TOCSY spectra of synthetic and recombinant ET-1. Spectra were recorded at 298 K in acetic acid-d3/H2O (40:60 v/v) at pH 2.5. (B) 1H–15N HSQC of uniformly labelled ET-1 recorded at 298 K in acetic acid-d3/H2O (40:60 v/v) at pH 2.5. Backbone assignments are as indicated.

for synthetic ET-1 to published 1H NMR assignments [15] and could conWrm their identities. Second, the spectra of both the synthetic and recombinant ET-1 were overlayed. Since overlapping 1H resonances were observed, we concluded that the conformational state of recombinant ET-1 is identical to synthetic ET-1. The proportion of the overlapping conformation as evaluated from the ratio of main to side resonances was at least 80% for both synthetic and recombinant ET-1. Since synthetic ET-1 previously showed high aYnity to the ETBR in biochemical displacement assays (IC50 D 1.7 £ 10¡10 M, [16]; IC50 D 3 £ 10¡10M; our own unpublished data), we conclude from the identity of

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259

H NMR spectra that biochemically active ET-1 was obtained using our protocol. To carry out assignment of resonances to the primary sequence, we conducted heteronuclear NMR experiments: The assignment of backbone (C, HN, N) and side chain C resonances (Table 2) was inferred from 1H–15N HSQC (Fig. 3B) and additional HNCACB, HN(CO)CACB triple resonance experiments. The Wngerprint region of 1H–15N HSQC spectrum showed a good dispersion of the amideproton resonances in the region from 7.0 to 10 ppm. Therefore, we concluded that also the uniformly 13C/15N labelled ET-1 was present in the folded conformation.

(51 kDa). In addition, the chromatography step requires preparation of a hydropathic peptide complementary to bigET-1 and immobilization of this peptide to the aYnity column. The method does not enable use of standard puriWcation techniques employed routinely in many laboratories. The method described by Becker et al. [7] consisted of expression of an AP-bigET-1 fusion, solubilization of inclusion bodies, two-steps of enzyme digestions with collagenase and dipeptidylpeptidase IV each followed by RPFPLC puriWcation to release bigET-1. Similar to Fassina et al. [8], the size of the AP-bigET-1 fusion is large (52 kDa), and the overall process costs are substantial (two-step enzyme digestion, fermentation). This protocol yielded 1.1 mg/L bigET-1, which may be further processed to ET-1. Interestingly, with this method two disulWde conformers of bigET-1 in a molar ratio of 3:1 could be separated using RP-FPLC. For chemically synthesized ET-1, also two conformers were separated with HPLC in the same ratio [17]. In comparison to these protocols, our method shows the following advantages: ET-1 was produced as minor partner of a rather small (18.1 kDa) Trx fusion protein. The puriWcation and proteolysis strategies are straightforward and cost-eVective. From 1 L of bacterial culture, we puriWed up to 5 mg ET-1 to homogeneity with a high overall process yield (up to 57%). In conclusion, this work describes the application of a thioredoxin fusion strategy for production, aYnity puriWcation and isotope labelling of ET-1. Isotopically labelled peptides aid in resolving peptide resonances in NMR spectra, and are required to analyze the interaction of small peptides with proteins (e.g., to resolve the structure of a receptor-bound ligand; [4]) using NMR. The expression strategy reported here should prove to be valuable for the recombinant production of isotopically labelled short peptides in general.

Discussion

Acknowledgments

Substantial experimental eVort has been expended to establish a recombinant expression system for ET-1. Previous studies employed the following strategies: The production method described by Yasufuku et al. [9] consisted of expression of a -galactosidase-bigET-1 fusion, solubilization of inclusion bodies, single-step puriWcation and sequential trypsin and pepsin digestions to release at Wrst bigET-1 and then ET-1. Finally, ET-1 was puriWed from the enzymatic digestions by RP-HPLC. Although this method is cost-eYcient and straightforward, only 0.6 mg/L ET-1 were obtained, mainly due to low expression level of the fusion protein. The method described by Fassina et al. [8] consisted of expression of a MBP-bigET-1 fusion, trypsin treatment to release bigET-1, a single-step aYnity chromatography, cleavage of bigET-1 with -chymotrypsin to release ET-1 and puriWcation of ET-1 by RP-HPLC. Although this method is relative eYcient (3.1 mg/l ET-1), the overall process yield is low due to the large size of the fusion protein

We gratefully acknowledge H. Lerch for providing MS data and Dr. C. Horn for helpful discussions on and very critical reading of this manuscript. J.R.P. acknowledges the FMP-Berlin and FAPERJ and CNPq agencies (Brazil) for funding. This project was Wnancially supported by the DFG (Sonderforschungsbereich 449).

Table 2 Resonance assignments for uniformly 13C/15N labelled ET-1 Residue 1

Cys Ser2 Cys3 Ser4 Ser5 Leu6 Met7 Asp8 Lys9 Glu10 Cys11 Val12 Tyr13 Phe14 Cys15 His16 Leu17 Asp18 Ile19 Ile20 Trp21

Chemical shift (ppm) C

C

NH

N

52.7 54.9 50.9 57.2 54.7 53.5 52.8 49.6 57 56.1 57.1 63.8 58.1 58.4 52.5 53.9 52.9 50.4 58.2 58.1 53.6

36.4 62.3 39.7 60.6 61.8 38.7 29.6 35.3 29.7 25.9 38.0 29.1 35.4 36.6 36.5 24.9 39.7 35.3 36.2 36 27

8.9 8.3 9.0 7.8 8.6 8.2 7.6 8.3 8.5 7.6 8.2 8.0 8.4 8.7 8.1 7.9 8.3 7.8 7.9 8.0

120.0 119.2 118.7 113.7 124.1 115.2 118.5 123.9 117.3 118.6 120.3 119.5 119.3 115.1 117.4 119.2 118.4 119.8 123.1 126.3

1

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