Cellfree expression of disulfidecontaining ... - Wiley Online Library

Cell-free expression of disulfide-containing eukaryotic proteins for structural biology Erich Michel and Kurt Wu¨thrich Institute of Molecular Biology and Biophysics, ETH Zurich, Switzerland

Keywords batch-mode cell-free protein expression; disulfide bond formation; Escherichia coli S30 cell extract; NMR spectroscopy; stable-isotope labeling Correspondence K. Wu¨thrich, Institute of Molecular Biology and Biophysics, ETH Zu¨rich, CH-8093 Zu¨rich, Switzerland Fax: +41 44 633 1151 Tel: +41 44 633 2473 E-mail: [email protected] (Received 26 April 2012, revised 8 June 2012, accepted 3 July 2012)

We describe Escherichia coli based cell-free production of milligram quantities of eukaryotic proteins containing native disulfide bonds. Using a previously described expression system, we systematically investigated the influence of redox potential variation in the reaction mixture and the impact of adding disulfide bond catalysts on soluble protein production. It is then shown that the optimized reaction conditions for native disulfide bond formation can be combined with the use of N-terminal fusion constructs with the GB1 domain for increased expression yields. The resulting cell-free system is suitable for stable-isotope labeling and does not require chemical pretreatment of the cell extract to stabilize the redox potential. For the human doppel protein, the mouse doppel protein and mouse interleukin-22 we obtained 0.3–0.7 mg of purified native protein per milliliter of reaction mixture. Formation of disulfide bonds was validated using the Ellman assay, and native folding of the three proteins was monitored by NMR and CD spectroscopy.

doi:10.1111/j.1742-4658.2012.08697.x Structured digital abstract l mIL22 and mIL22 bind by nuclear magnetic resonance (View interaction)

Introduction Proteins containing disulfide bonds are typically exported from the cytoplasm [1,2] and function in cell signaling, signal transduction, immune response and other vital processes [3]. Notwithstanding the keen scientific interest in this class of eukaryotic proteins, preparation of milligram amounts for structural and functional studies remains a challenge, mainly for the following reasons. (a) Eukaryotic expression systems tend to be economically unfavorable due to low protein yields and high cell culture costs [4]. (b) In expression hosts such as Escherichia coli the formation of the native disulfide bonds is difficult and often also results in low yields of folded protein [2,5],

especially if the protein needs to be refolded from inclusion bodies [6,7]. (c) Periplasmic expression provides both an oxidative environment and enzymes which facilitate correct disulfide bond formation, but the translocation capacity for proteins targeted for the periplasm is limited, which often results in low yields and accumulation of non-native protein in the cytoplasm [8]. Cell-free expression provides an alternative strategy for production of disulfide-containing proteins, by direct supplementation of the reaction mixture with catalysts that support disulfide bond formation and native folding of the translated proteins [9]. However,

Abbreviations 2-ME, b-mercaptoethanol; DsbA, thiol : disulfide interchange protein DsbA; DsbC, thiol : disulfide interchange protein DsbC; DTNB, 5,5¢-dithiobis-2-nitrobenzoic acid; DTT, dithiothreitol; GB1, B1 domain of protein G from Streptococcus sp.; GSH, reduced glutathione; GSSG, oxidized glutathione; hDpl(24–152), human doppel protein construct with residues 24–152; HSQC, heteronuclear single quantum coherence; mDpl(24–155), murine doppel protein construct with residues 24–155; mIL22(34–179), murine interleukin-22 construct with residues 34–179; OmpX, outer membrane protein X; PDI, protein disulfide isomerase.

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E. Michel and K. Wu¨thrich

this approach also meets with challenging obstacles. Thus, systems based on eukaryotic cell extracts typically provide microgram amounts of protein per milliliter of reaction mixture [10,11], and the extract preparation is work intensive and expensive [12,13]. E. coli-based cell-free expression systems provide higher intrinsic productivity, but adaptation for disulfide bond formation tends to reduce the yield to the microgram scale of native protein per milliliter of reaction mixture [9,14,15]. Chemical pretreatment of the cell extract in E. coli-based systems for production of native disulfide-containing proteins tends to lower translation activity [14,15], and reactivation of central metabolic pathways in the cell extract to provide energy for translation [16,17] prevents efficient stable-isotope labeling of proteins for NMR studies [18–20]. Here we describe the production of milligram amounts of disulfide-containing proteins per milliliter of reaction mixture by a novel optimization of an E. coli-based batch-mode cell-free expression system [21]. In addition to a high yield of natively folded protein, a key advantage of this system is the use of eukaryotic creatine phosphate based energy regeneration that avoids central metabolic pathways of E. coli. This is a critical advantage for NMR spectroscopy, since phosphoenolpyruvate-based energy regeneration produces intermediates of amino acid biosynthesis that interfere with amino acid selective stable-isotope labeling [16,17]. ATP regeneration based on creatine phosphate further avoids reduction of oxidized glutathione (GSSG) during cell-free expression, which results in a stable redox potential without requiring chemical pretreatment of the cell extracts. The system also avoids the use of glutamate buffer, which would result in isotope scrambling and dilution [16]. Use of the system is illustrated with the human prion-like doppel protein, hDpl(24–152), the mouse prion-like doppel protein, mDpl(24–155), and mouse interleukin-22, mIL22(34– 179). All three proteins contain two non-consecutive disulfide bonds and could not be produced in soluble form by recombinant expression in E. coli [22–24]. For hDpl(24–152) and mDpl(24–155), NMR resonance assignments are available, so that the native fold of the cell-free product could be validated by comparison with literature data [23,24].

Results and Discussion There are two distinct aspects of the procedure introduced here. First, multiple components of the expression mixture were individually screened for optimized formation of native disulfide bonds. Second, it is

Cell-free expression of disulfide-containing proteins

shown that these optimized solution conditions can be combined with GB1 (the B1 domain of protein G from Streptococcus sp.) fusion for enhanced expression [21]. The success of this combination is linked to subtle details in preparing the reaction mixture. Activity of cell-free protein synthesis under oxidizing conditions The translation machinery in the starting expression system for this project [21] was derived from the reducing environment of the E. coli cytoplasm. We therefore first determined the protein synthesis yields in this system under increasingly oxidizing conditions. Smallscale expression of the membrane protein OmpX, which is exclusively produced in the insoluble fraction, was performed in reaction mixtures supplemented with variable ratios of reduced glutathione (GSH) and GSSG (Fig. 1). SDS ⁄ PAGE analysis of the insoluble expression product indicated that the intrinsic protein synthesis is not inhibited by addition of up to 10 mM GSSG, even in the absence of GSH (Fig. 1). At a level of 20 mM GSSG the production of OmpX decreased significantly, and at 30 mM GSSG the cell-free system no longer yielded measurable synthesis of OmpX. These results can be rationalized by the observation that protein components of the cell-free system precipitated at GSSG concentrations above 10 mM, and so they could be detected in the insoluble protein fraction. Addition of up to 20 mM GSH in the absence of GSSG did not measurably affect the translation efficiency. N - 2 2 2 - 0.1 0.5 2

2 2 1 - - - - GSH [mM] 5 10 10 10 20 30 40 GSSG [mM]

OmpX

Fig. 1. Impact of redox conditions on the translational activity of the E. coli based cell-free expression system [21] that was used as the starting point for the present study. Cell-free expression of the test protein OmpX in pIVEX2.4 was conducted for 2 h at 30 C in the presence of the indicated ratios of GSH and GSSG. After the reaction, the insoluble protein fraction containing OmpX was collected and analyzed by SDS ⁄ PAGE. Each lane corresponds to 3.5 lL of reaction mixture. N indicates the negative control reaction without template DNA.


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Fine adjustment of redox conditions for disulfide bond formation in the cell-free system To determine appropriate redox conditions that favor disulfide bond formation and native folding without sacrificing translation efficiency, we investigated soluble production of hDpl(24–152)-pIVEX2.4 in cell-free reaction media supplemented with various ratios of GSH and GSSG. Western blot analysis of the reaction supernatant revealed that the highest soluble expression of hDpl(24–152) was obtained in the presence of 2 mM GSH and 5–10 mM GSSG (Fig. 2A). Since the reaction mixture also contains 2.1 mM dithiothreitol (DTT), 2.1 mM b-mercaptoethanol (2-ME) and 1.5 mM cysteine (Table 1), high soluble expression was thus obtained in the presence of about 10 mM free sulfhydryl groups and 5–10 mM GSSG. Supplementing the cell-free expression system with disulfide bond catalysts SDS ⁄ PAGE comparison of both soluble and insoluble yields of hDpl(24–152) revealed that only about 5% (8 lgÆmL)1) of the total hDpl produced (160 lgÆmL)1) was obtained in soluble form at the aforementioned optimal redox conditions. In an attempt to increase soluble production of hDpl(24–152), the reaction mixture with redox conditions fixed at 2 mM GSH and 5 mM GSSG was supplemented with various concentrations of thiol : disulfide interchange protein DsbC (DsbC) and protein disulfide isomerase (PDI). Western blot analysis showed that a supplement of 10 lM DsbC or A

N -

-

2 2 2 0.1 0.5 1

2 2

2 5

2 2 1 - GSH [mM] 10 20 20 20 GSSG [mM]

PDI significantly increased soluble production of hDpl(24–152) (Fig. 2B). Thereby more than 50% (80 lgÆmL)1) of the synthesized hDpl(24–152) was obtained in soluble form. The conditions thus established for optimal expression of hDpl(24–152) could be carried over to GB1–mDpl(24–155) (Fig. 2C). For mIL22(34–179), SDS ⁄ PAGE analysis indicated that, in the absence of enzymatic disulfide bond catalysts, maximal production of soluble protein was achieved after addition of 2 mM GSH and 5–10 mM GSSG. Addition of 5 lM DsbC further increased production of the soluble protein 2.5-fold (Fig. 2D), and more than 80% (0.2 mgÆmL)1) of the total synthesized mIL22(34–179) (0.25 mgÆmL)1) was obtained in soluble form. Since maximum soluble yields were obtained with 5 lM DsbC, there is an indication that the native arrangement of disulfide bonds is more readily achieved for mIL22(34–179) than for hDpl(24– 152) or for mDpl(24–155). The disulfide bond oxidase thiol : disulfide interchange protein DsbA (DsbA) has previously been shown to enhance native folding of proteins in vitro by enhancing disulfide bond formation during the folding process [25]. We therefore also investigated the effects of adding up to 25 lM DsbA, prepared as described in Doc. S3, to a reaction mixture that contained also 2 mM GSH, 5 mM GSSG and 10 lM DsbC. Under these conditions the addition of DsbA did not increase the soluble yield of hDpl(24–152). Similarly, addition of DsbA in the absence of DsbC did not enhance soluble expression, and we did not further pursue experimenting with DsbA. Overall, both the prokaryotic B

N -

-

1

1 -

- 2.5 2.5 - 5

5 - 10 - 20 PDI [μ μM] - 10 - 20 - DsbC [μM] hDpl (24–152)

hDpl (24–152)

C

N -

-

D

10 25 10 25 10 25 10 25 DsbC [μM] 2 2 2 2 2 2 GSH [mM] 2 2 5 5 10 10 GSSG [mM]

- - 2 2 2 0.1 0.5 2

2 5

N - - 5 5 5 2 - 2 2 2 10 - 0.1 0.5 2

5 2 5

5 DsbC [μM] 2 GSH [mM] 10 GSSG [mM]

kDa

GB1-mDpl (24–155)

25 17

mIL22 (34–179)

Fig. 2. Influence of variable redox potentials and disulfide bond isomerase concentrations on the cell-free expression of three target proteins containing disulfide bonds. Shown are western blot (A, B, C) and SDS ⁄ PAGE (D) analyses of the soluble proteins obtained after cell-free expression during 2.5 h at 30 C using either the pIVEX2.4 (A, B, D) or the pCFX1 (C) vector. For each lane in (A), (C) and (D) the applied sample volume corresponds to 2.5 lL of reaction mixture, and in (B) to 1.5 lL. (A) Soluble expression levels of hDpl(24–152) at various redox potentials achieved by mixing different ratios of GSH and GSSG. (B) Reaction mixtures for expression of hDpl(24–152) that contained 2 mM GSH and 5 mM GSSG were supplemented with increasing concentrations of the disulfide bond isomerases DsbC or PDI. (C) Soluble cell-free expression of GB1–mDpl(24–155) at variable levels of the redox potential and DsbC concentration. (D) Effects of variable redox potentials and DsbC concentrations on the soluble production of mIL22(34–179). Arrows indicate the bands corresponding to the target protein, and N identifies the lane of the negative control reaction without template plasmid.

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Table 1. Composition of the cell-free reaction mixture. Component

Concentration

KOAc Creatine phosphate HEPES-KOH Mg(OAc)2 NaN3 PEG-8000 2-ME DTT ATP GTP CTP UTP cAMPÆNa+ Folinic acidÆCa2+ E. coli tRNA Creatine kinase Target plasmid T7 RNA polymerase Amino acids (each) S30 cell extract

217 mM 80 mM 58 mM 11 mM 3.8 mM 3.25% (w ⁄ v) 2.1 mM 2.1 mM 1.2 mM 0.86 mM 0.86 mM 0.86 mM 0.64 mM 68 lM 175 lgÆmL)1 5.8 lM 10 lgÆmL)1 65 nM 1.5 mM 30% (v ⁄ v)

DsbC and the eukaryotic PDI promoted expression of soluble target protein, indicating that they can be used interchangeably in the present expression system. Cell-free expression with S30 cell extract from E. coli Origami (DE3) cells E. coli strains containing mutations in the genes encoding thioredoxin reductase (trxB) and glutathione reductase (gor) have been shown to yield enhanced disulfide bond formation in the bacterial cytoplasm [26]. To investigate the potential of extracts from these strains for cell-free production of proteins containing disulfide bonds with the protocol optimized for BL21 (DE3) RIPL-Star, we prepared S30 extract from the E. coli Origami (DE3) strain (Novagen, Darmstadt, Germany). We then compared cell-free production of mIL22(34–179) using S30 extracts prepared from either E. coli Origami (DE3) or E. coli BL21 (DE3) RIPLStar cells, which were supplemented with various ratios of GSH and GSSG. Analysis by SDS ⁄ PAGE indicated very low expression levels of mIL22(34–179) in reactions with S30 extract from Origami cells, and we did not further investigate this extract. N-terminal GB1 fusion for increased expression yields We recently showed that N-terminal fusion constructs with the GB1 domain have increased yields of cell-free expression of human proteins [21]. Here we explore the

use of GB1 fusion with the aforementioned cell-free systems that have been optimized for disulfide-containing proteins. Overall, GB1 fusion of the target proteins was thus found to affect neither the optimal GSH ⁄ GSSG ratio nor the optimal concentration of disulfide bond isomerase required for native folding. Thus 10 mL cell-free reactions of hDpl(24–152) with (pCFX1) or without (pIVEX2.4) fusion with the GB1 solubility domain were carried out at optimum conditions for disulfide bond formation, i.e. with 2 mM GSH, 5 mM GSSG and 10 lM DsbC. After purification, UV absorption at 280 nm indicated yields of 4.2 mg soluble hDpl(24–152) from pCFX1 and 0.8 mg from pIVEX2.4. The 5-fold increase for the fusion construct with the GB1 domain is probably due to both increased total production of the protein by enhanced translation efficiency [21] and increased solubility [27]. SDS ⁄ PAGE showed that the yield of 4.2 mg corresponds to 75% of the total hDpl(24–152) expression (5.6 mg), compared with 2% of the total synthesized protein in the absence of disulfide-forming additives. In further assays, 10 mL reactions of mIL22(34–179) in either pIVEX2.4 or pCFX3 were carried out using [u-15N]-amino acids at optimum conditions for mIL22(34–179), i.e. with addition of 2 mM GSH, 10 mM GSSG and 5 lM DsbC. We obtained 2.0 mg of mIL22(34–179) from expression in pIVEX2.4 and 6.6 mg from expression in pCFX3, where SDS ⁄ PAGE indicated that more than 90% (6.6 mg) of the total produced N-terminal fusion construct with the GB1 domain (7.3 mg) was soluble. In the absence of disulfide-promoting additives, only 5% of the expressed GB1–mIL22 fusion protein was soluble. Finally, a 10 mL cell-free reaction using [u-15N]-labeled amino acids in pCFX1 with addition of 2mM GSH, 5 mM GSSG and 10 lM DsbC yielded 3.0 mg of purified mDpl(24–155), corresponding to 50% of the total expressed protein (6 mg). The lower yield of soluble protein, compared with hDpl(24–152), seems to reflect increased difficulty in producing the mouse protein, as was previously observed with expression in E. coli cell cultures [23,24]. The yield of soluble GB1–mDpl fusion protein without disulfide-promoting additives was 2% of the total synthesized protein. Structural validation of the disulfide-containing proteins from cell-free production and conclusions The free sulfhydryl content of the three proteins studied here was determined with the Ellman assay under denaturing conditions, as described in the Materials and methods section. There was no evidence of


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measurable free sulfhydryl content, implying that the disulfide bonds had been formed successfully. To obtain evidence that the proteins produced by cell-free expression were natively folded, we recorded 2D [15N, 1H] heteronuclear single quantum coherence (HSQC) NMR spectra. For cell-free produced hDpl(24–152) and mDpl(25–155), comparison with the available backbone amide resonance assignments [23,24] indicated that the native fold was obtained (Fig. 3). This includes that hDpl(24–152) expressed either free or as a fusion construct with the GB1 domain adopted the native fold (Fig. 3A,B). The spectra also contain NMR signals from the N-terminal purification and solubility tags. Human interleukin-22 had been shown to form dimers in solution even at micromolar concentrations [28]. It appears that mIL22(34–179) is also dimeric in solution, since the 2D [15N, 1H]-HSQC spectrum contains a lesser number of signals than expected from the number of amide groups in the protein, with most residues represented by weak peaks (Fig. 4A). It can nonetheless be recognized that the NMR spectrum corresponds to a folded globular protein. For the denatured protein in urea, the 2D [15N, 1H]-HSQC

A

spectrum contained a number of amide resonances which, within the accuracy of the peak count, agreed closely with the number expected from the primary structure (Fig. 4B). The cell-free produced mIL22(34– 179) showed a far-UV CD spectrum typical of a protein with a high content of helical secondary structure (Fig. 5), and biological activity of the produced protein was verified using IL-22-deficient mice in the laboratory of B. Becher [29]. Overall, the present documentation of native folding of disulfide-containing eukaryotic proteins produced in milligram quantities in a cell-free system validates the cell-free expression protocol used, which included adjusting an appropriate redox potential by addition of small concentrations of GSH and GSSG to S30 cell extract from the E. coli BL21 (DE3) RIPL-Star strain and supplementing this mixture with a disulfide bond isomerase to facilitate the correct arrangement of disulfide bonds. It is worth noting that the best results were obtained in the presence of 10 mM sulfhydryl groups and 5–10 mM GSSG, which corresponds closely to the redox conditions in the endoplasmatic reticulum [30], where disulfide bonds are typically formed [2]. The renewed observation of significantly increased soluble

B

ω1(15N) [p.p.m.]

C

110

120

130 10

8

6

10

8

6

10

8

ω2(1H) [p.p.m.]

Fig. 3. 2D [15N, 1H]-HSQC spectra of hDpl(24–152) and mDpl(24–155) prepared by cell-free expression with 15N-labeled amino acids in reaction mixtures containing 2 mM GSH, 7.5 mM GSSG and 10 lM DsbC. (A) 200 lM [u-15N]-hDpl(24–152) produced from two 10 mL reactions using the pIVEX2.4 vector. (B) 275 lM [u-15N]-GB1–hDpl(24–152) prepared from a 10 mL cell-free reaction using the pCFX1 vector. The additional signals originating from the GB1 domain [27] can be readily recognized. (C) 140 lM [u-15N]-mDpl(24–155) after thrombin cleavage to remove the N-terminal GB1 solubility domain. The protein was synthesized in a 10 mL batch-mode cell-free reaction using the pCFX1 vector. All samples contained 20 mM sodium acetate at pH 5.2, 100 lM EDTA, 10 lM sodium azide and 5% D2O. The spectra were recorded at a 1 H resonance frequency of 750 MHz with T = 20 C. Red crosses in (A) and (B) show the published peak positions for hDpl(24–152) (BMRB 5145), and those in (C) the peak positions for mDpl(26–157) (BMRB 4938).

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A

B 110

ω1(15N) [p.p.m.] 120

9

8

7

9

8

ω2(1H) [p.p.m.]

Fig. 4. 2D [15N, 1H]-HSQC spectra for (A) native and (B) urea-unfolded [u-15N]-mIL22(34–179) expressed with the vector pIVEX2.4 in a 10 mL cell-free reaction mixture supplemented with 2 mM GSH, 10 mM GSSG and 5 lM DsbC. In (B) the protein was denatured in 8 M urea and 10 mM DTT. The spectra were recorded at a 1H resonance frequency of 750 MHz and at T = 37 C.

enables the production of stable-isotope-labeled proteins. It further avoids reduction of GSSG during the cell-free reaction and thus eliminates the requirement for chemical pretreatment of the S30 extract. These results set our expression system apart from previously described cell-free systems. These either provided yields of < 50 lg native protein per milliliter of reaction mixture [9–11,14] or were based on energy regeneration with phosphoenolpyruvate [14,16,17], which is incompatible with stable-isotope labeling and requires chemical pretreatment of the cell extract to stabilize the redox potential [14–16].

[Θ] x 10–3 (deg·cm2·dmol–1) 60

40

20

0

–20

Materials and methods Cloning of target genes 200

220

240

Wavelength (nm)

Fig. 5. Far-UV CD spectrum at 20 C of mIL22(34–179) produced by cell-free synthesis. The sample contained 15 lM mIL22(34–179), 3 mM sodium phosphate at pH 7.0, 34 mM sodium chloride and 1.1 mM potassium chloride.

expression of N-terminal fusion constructs of the target protein with the GB1 domain [21] indicates that this approach might be quite widely applicable to obtain increased yields in cell-free expression. With regard to using this expression system in structural biology, it is of interest that energy regeneration is based on creatine phosphate, which does not activate metabolic pathways in the cell extract and therefore

The gene encoding residues 24–152 of the human doppel protein hDpl(24–152) was amplified by PCR (HotStar Taq Master Mix Kit, Qiagen, Hombrechtikon, Switzerland) from hDplpRSETA [24], using GGA ATT CCA TAT GGT CCA GAC GAG GGG CAT C and CGC GGA TCC TTA GCC CCT CTC CAA CCA AAA C as 5¢- and 3¢-oligonucleotide primers, respectively. The murine doppel protein mDpl(24–155) was amplified from mDpl-pRSETA [31], using GGA ATT CCA TAT GGT CAA GGC AAG GGG CAT AAA G and CGC GGA TCC TTA TCC CCT TTC CAG CCA GAA ATC as 5¢- and 3¢-oligonucleotide primers, respectively. The PCR products were cloned into pIVEX2.4 (Roche, Rotkreuz, Switzerland) and pCFX1 [21] using the NdeI and BamHI restriction sites. Mouse interleukin-22, mIL(34–179), was subcloned from mIL22-pIVEX2.4 [29] into pCFX3 [21] using the NdeI and BamHI restriction sites. The gene encoding the E. coli outer membrane protein X (OmpX) was subcloned from pET3b-


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OmpX [32] into pIVEX2.4, using the NdeI and BamHI restriction sites. All constructs were verified by sequencing (Microsynth, Balgach, Switzerland), and the plasmids for cellfree expression were prepared using a plasmid maxi prep kit (Macherey-Nagel, Oensingen, Switzerland).

Cell-free protein expression All solutions other than the culture media were prepared using diethylpyrocarbonate-treated water. S30 cell extracts were prepared from 9 L of cultures in phosphate ⁄ yeast extract ⁄ glucose medium [5.6 gÆL)1 KH2PO4, 28.9 gÆL)1 K2HPO4, 10 gÆL)1 yeast extract, 1% (w ⁄ v) glucose] of either E. coli BL21 (DE3) RIPL-Star [21] or E. coli Origami (DE3) (Novagen, Darmstadt, Germany) cells, following a previously described protocol [21]. The S30 cell extract was stored in 500 lL aliquots at )80 C. Unless indicated otherwise, all other reagents required for preparing the reaction mixture were stored at )20 C. The standard cellfree reaction mixture contained 58 mM HEPES ⁄ KOH at pH 8.2, 217 mM potassium acetate, 175 lgÆmL)1 E. coli tRNA (Sigma, Buchs, Switzerland), 3.25% (v ⁄ v) PEG-8000 (Fluka, Buchs, Switzerland), 11 mM magnesium acetate (Applichem, Darmstadt, Germany), 2.1 mM DTT (Applichem, Darmstadt, Germany), 2.1 mM 2-ME (Applichem, Darmstadt, Germany), 1.2 mM ATP (Applichem, Darmstadt, Germany), 0.86 mM each of GTP (Fluka, Buchs, Switzerland), CTP and UTP (both Applichem, Darmstadt, Germany), 80 mM creatine phosphate (Sigma, Buchs, Switzerland), 5.8 lM creatine kinase from rabbit muscle (Roche, Rotkreuz, Switzerland), 3.8 mM sodium azide (Fluka, Buchs, Switzerland), 1.5 mM of each of the 20 proteinogenic amino acids (Spectra Stable Isotopes, Andover, MA, USA), 68 lM folinic acid (Sigma, Buchs, Switzerland), 640 lM cAMP (Sigma, Buchs, Switzerland), 0.65 lM T7 RNA polymerase, 10 lgÆmL)1 template plasmid and 30% (v ⁄ v) S30 cell extract. Reactions were conducted in batch mode for 2.5 h at 30 C in a Thermomixer Comfort (Eppendorf, Basel, Switzerland) with gentle shaking. Analytical-scale reactions (50 or 100 lL) were conducted in 1.5 mL tubes, and preparativescale expressions (10 mL) were carried out in 15 mL tubes. The products of the analytical-scale reactions were centrifuged for 3 min at 14 000 g, and the supernatant was analyzed by SDS ⁄ PAGE and western blotting, as previously described [21]. The products of preparative-scale reactions were centrifuged for 5 min at 4000 g, and the supernatant was used for purification, as described below. Cell-free expression with concomitant disulfide bond formation started with reaction mixtures prepared as described above. The disulfide bond isomerases DsbC or PDI, prepared as described in Docs S1 and S2, were then added to the reaction mixture. Finally, the redox conditions were adjusted by first adding GSH followed by addition of GSSG (Sigma, Buchs, Switzerland) and gentle mixing of the reaction solution.

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Purification of cell-free expression products Purification of proteins obtained by cell-free expression was performed at 4 C, using an Aekta prime FPLC system (Amersham, Glattbrugg, Switzerland) equipped with A280 and conductivity measurement devices. The cleared protein solutions obtained after centrifugation of preparative-scale cell-free reactions were applied to a 5 mL HisTrap HP column (GE Healthcare, Glattbrugg, Switzerland) in buffer A (see below). After washing with 10 column volumes of buffer A, the protein of interest was eluted in a 100 mL linear gradient of 30–500 mM imidazole in buffer A. For both the human and mouse doppel proteins, buffer A comprised 50 mM potassium phosphate at pH 7.2, 30 mM imidazole and 300 mM potassium chloride. For interleukin-22, the buffer composition was 50 mM sodium phosphate at pH 7.4, 30 mM imidazole and 500 mM sodium chloride. Collected fractions showing absorption at 280 nm were analyzed by SDS ⁄ PAGE. Proteolytic cleavage of the GB1 fusion domain using thrombin or tobacco etch virus protease was conducted as described previously [21].

Determination of free thiol content The free thiol content of the purified proteins was determined using the Ellman assay [33,34]. Various dilutions of purified protein with buffer EA [100 mM Tris ⁄ HCl at pH 7.8, 2 mM EDTA, 2% (w ⁄ v) SDS] were prepared in a total volume of 300 lL. For determination of a standard curve, 300 lL solutions containing 0, 1, 5, 10, 25, 50 and 100 lM GSH were prepared in buffer EA. Samples were then mixed with 10 lL of 10 mM 5,5¢-dithiobis-2-nitrobenzoic acid and incubated for 5 min at room temperature before recording the absorbance at 412 nm. All measurements were conducted as triplicates, and the free thiol content of the purified protein was determined by comparison with the aforementioned standard curve.

Circular dichroism (CD) spectroscopy Far-UV CD spectra were recorded on a JASCO J-715 spectropolarimeter (JASCO, Easton, MD, USA) at 20 C, using 1 mm QS quartz cuvettes (Hellma Analytics, Mu¨llheim, Germany) and scanning from 250 to 190 nm. The results were displayed as molar ellipticity Q with units of deg cm2Ædmol)1.

NMR sample preparation For each protein a 10 mL batch-mode cell-free reaction with 15N-labeled amino acids (Spectra Stable Isotopes, Andover, MA, USA) was set up, and the proteins were purified as described above and dialyzed overnight against 4 L of NMR buffer. For the mouse and human doppel



proteins the buffer contained 20 mM sodium acetate at pH 5.2, 100 lM EDTA and 10 lM sodium azide. For interleukin-22 the NMR buffer contained 6 mM sodium phosphate at pH 7.0, 68 mM sodium chloride and 2.2 mM potassium chloride. After dialysis, the protein solutions were concentrated to 500 lL using a 10 kDa Amicon Ultra-15 centricon (Millipore, Zug, Switzerland) and were supplemented with 5% (v ⁄ v) D2O before transfer into a 5TA NMR sample tube (Armar Chemicals, Do¨ttingen, Switzerland).

NMR spectroscopy NMR spectra were recorded on a Bruker DRX-750 spectrometer equipped with a room-temperature triple resonance probe with shielded z-gradient coils. Spectra were recorded at either 20 or 37 C, depending on the protein, and were processed with TOPSPIN 2.0 (Bruker-Biospin, Fa¨llanden, Switzerland) followed by analysis using the program CARA [35].


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Acknowledgements We thank Fabian Mu¨ller for help with cell extract preparations, Dr Lars Ellgaard for providing the pET12a-PDI plasmid, Dr Rudi Glockshuber for providing the plasmids pDsbC and pASK40-DsbA, and the ETH Zu¨rich and the Swiss National Science Foundation for financial support through the National Center of Competence in Research (NCCR) Structural Biology.

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Supporting information The following supplementary material is available: Doc. 1. Preparation of DsbC. Doc. 2. Preparation of human PDI. Doc. 3. Preparation of DsbA. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
