Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of γ-Gliadin

Protein Expression and Purification 46 (2006) 358–366 www.elsevier.com/locate/yprep

Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of c-Gliadin Claudia G. Benitez-Cardoza a,*, He´le`ne Rogniaux b, Yves Popineau b, Jacques Gue´guen b a

b

Programa Institucional en Biomedicina Molecular ENMyH-IPN, Guillermo Massieu Helguera No. 239 Fraccionamiento ‘‘La Escalera Ticoman’’ CP 07320, Me´xico DF, Mexico Unite´ de Recherche sur les Biopolyme`res, leurs Interactions et leurs Assemblages Rue de la, Ge´raudie`re, B.P. 71627/44316 Nantes Cedex 03, France Received 7 July 2005, and in revised form 19 August 2005 Available online 20 September 2005

Abstract The gene encoding the repetitive domain located in the N-terminal half of c-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of c-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypetides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.  2005 Elsevier Inc. All rights reserved. Keywords: c-Gliadin; Repetitive domain; Circular dichroism; Thioredoxin; Wheat storage proteins

The wheat seed storage proteins are a major source of protein in the human diet and are responsible for the properties of wheat doughs that allow a wide range of food products. Gliadins and glutenins (prolamins) are the major storage proteins that accumulate in wheat endosperm cells during seed development. Prolamins are initially deposited in ER-derived protein bodies, then they form a protein matrix between starch granules when the grain is drying [1]. Gliadins are monomeric but glutenins consist of very large disulfide-linked polymers made up of high molecular weight and low molecular weight subunits. Gliadins can be extracted from wheat flour or gluten in 70% aqueous alcohol. Three groups have been identified, a/b, c-, and

*

Corresponding author. Fax: +525557296300. E-mail address: [email protected] (C.G. Benitez-Cardoza).

1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.08.017

x-Gliadins, on the basis of their amino acid sequences and electrophoretic mobility at acid pH. All gliadins have a low content of basic amino acids, and almost all glutamic and aspartic residues are amidated. In c-Gliadins there are eight cysteins, forming four intramolecular disulfide bonds. The sequence of c-Gliadins contains a short N-terminal followed by a Gln-, Pro-rich repetitive domain, and a C-terminal domain. The Gln-, Pro-rich domain is found in all wheat prolamins. In c-Gliadins the repetitive domain contains up to 100–160 residues arranged as repeated sequences of one or two motifs composed of glutamine, proline, and aromatic aminoacids (phenylalanine or tyrosyne). While the N-terminal domain is a short region of about 5–14 aminoacid residues, the C-terminus domain is composed of some segments of di- or tri-glutamine, and unique sequences that hold all the cysteines and lysine residues, eight and three per molecule, respectively [2,3].

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By hydration and mixing, the wheat storage proteins form a complex, the gluten. The proportions of gliadins and glutenin, and the size distribution of the glutenin polymers are the determinants of the viscoelastic properties of wet gluten [3–5]. This is the basis of the rheological properties of the wheat flour doughs. According to the most recent findings, the repetitive domains of gliadins and glutenin subunits are especially involved in prolamin–prolamin interactions through numerous interchain hydrogen bonds, generated by the glutamine side chains [6,4]. Gliadins are implicated in coeliac disease, an autoimmune reaction triggered by some cereal proteins [7,8] and in food allergy to wheat [9,10]. Peptides from the repetitive domain of the gliadins are involved in both diseases [11–16]. Understanding the involvement of each of the domains of prolamins and their interactions in the protein bodies and matrix assembly of wheat endosperm, in the viscoelastic properties of wet gluten and their contributions in coeliac disease and in food allergy to wheat will help to improve technological and nutritional properties of wheat products. Because of the complexity of gene families, it was difficult to obtain single prolamins to be structurally, chemically, and immunologically characterized [17] before the advent of recombinant DNA technology. However, several structural features have been determined on prolamins isolated from wheat flour [18–22,4]. The secondary structure of c-44 gliadin and of polypeptides corresponding to its repetitive and non-repetitive domains derived from its enzymatic hydrolysis was characterized [23]. Several wheat prolamins, mainly glutenin subunits or glutenin domains, have been produced in Escherichia coli as insoluble aggregates or ‘‘inclusion bodies’’ [13,24–32]. This allowed to analyze in details the structure and physicochemical properties of the N-term and repetitive domains of the HMW glutenin subunits [25,26,29,30,33,34]. Synthetic genes encoding periodic polypeptides modelled on a consensus repeated sequence of wheat gliadins were designed and expressed in E. coli. [35,36]. Besides, cereal prolamins have also been expressed in eukaryotic systems, such as yeast [37–39] transgenic plants [40–45], and Xenopus oocytes [46]. The thioredoxin fusion system (pET system) has been successfully used to produce soluble target proteins which are otherwise insoluble in E. coli. For example, maize c-zeine had been cloned and their N- and C-terminus domains expressed in E. coli as a thioredoxin fusion resulting in enhanced solubility mainly of the C-terminus domain of the fusion protein when compared with the C-terminus domain without thioredoxin [47]. In this work, we report the sub-cloning, bacterial expression, and purification of the repetitive N-terminus domain of c-Gliadin from wheat endosperm, as thioredoxin fusion protein. To our knowledge, this is the first report of the expression of a repetitive N-terminal domain of c-Gliadin as a soluble protein in enterobacteria. The secondary structure of the native and thermally treated polypeptide was characterized, by circular dichroism (CD) spectroscopy.

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This kind of work is needed to obtain highly purified repetitive domains of prolamins, to further structural and chemical characterization. For example: it could be interesting to study the assembly of the storage proteins in the developing wheat grain. Materials and methods The plasmid containing the c-Gliadin gene was kindly provided by P.R. Shewry (Rothamsted Research, Rothamsted, UK). The protein sequence was that reported by Bartels et al. [48]. The sub-cloning was made using the vector pET102 (Invitrogen) This vector expresses fusion proteins of thioredoxin, that can be removed after protein purification using enterokinase. The thioredoxin expressed by this vector has been mutated to contain a metal binding domain and is termed ‘‘HisPatch thioredoxin.’’ To create a metal binding domain in the thioredoxin protein, the glutamate residues at position 32 and the glutamine residue at position 64 were mutated to histidine residues. When His-Patch thioredoxin folds, the histidines at positions 32 and 64 interact with a native histidine at position 8 to form a ‘‘patch.’’ This histidine patch has been shown to have high affinity for divalent cations [49]. In addition, an extra 6-His tag flanked by the residues Asp-Pro was introduced at the N-terminus of the proteins. The residues Asp-Pro; are known to be labile to acid cleavage. This provides an alternative way for the separation of the recombinant polypeptide from the thioredoxin and tag. It is worth to mention that residues 5 and 6 in the sequence of c-Gliadin are Asp-Pro, nevertheless an extra pair AspPro was introduced in our construct to make certain that the 6-His tag was removed from the polypeptide, in case the pair Asp-Pro was not fully accessible for acid cleavage. The complete sequence of the fusion protein translated from the DNA sequence is shown in Fig. 1. A repetitive polypeptide was prepared from native c-Gliadin by chymotryptic hydrolyis as described before [23]. Amplification and subcloning of genes Oligonucleotide primers (Invitrogen) were designed for amplification of the N-terminal-repetitive domain half of c-Gliadin gene coding for mature protein without leader sequence. Specific primers had the sequences as follows: sense primer (5 0 -CACCCATCATCACCATCACCATGA TCCCAATATGCAGGTCGACCCTA-3); and antisense primer (5 0 -CATGGATCCTTAGCCAATGAACGGTGG TTGTTGTTGGGGGA-3 0 ). For polymerase chain reaction (PCR)1 amplification, about 100 ng of cDNA was 1

Abbreviations used: NRG, N-terminal-repetitive domain of c-Gliadin; TRX-NRG, thioredoxin-N-terminal-repetitive domain of c-Gliadin fusion protein; Tris, Tris((hydroxy-methyl)aminomethane); GuCl, guanidinium chloride; PCR, polymerase chain reaction; SDS/PAGE, sodium duodecyl sulphate/polyacrylamide gel electrophoresis.

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Acid cleavable sites Site 1

Site 2

Site 3

MGSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDEIADEYQG KLTVAKLNIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLD ANLAGSGSGDDDDKLGIDPFTHHHHHHDPNMQVDPSSQVQWPQQQPVPQPHQP FSQQPQQTFPQPQQTFPHQPQQQFPQPQQPQQQFLQPQQPFPQQPQQPYPQQP QQPFPQTQQPQQLFPQSQQPQQQFSQPQQQFPQPQQPQQSFPQQQPPFIG Fig. 1. Thioredoxin-N-terminal-repetitive domain of c-Gliadin fusion protein sequence. N-terminal-repetitive domain of c-Gliadin domain is underlined. The acid-labile sites are shown in bold, and signalled by arrows showing the three Asp-Pro couples. Enterokinase site is italicised.

used in a reaction mixture containing 0.3 mM each dNTP (Amersham Pharmacia Biotech), 300 nM each primer, 1 mM MgSO4, 1.25 U of Platinum Pfx-DNA polymerase (Invitrogen). An initial denaturation step for 3 min at 94 C was followed by 30 cycles of denaturation, annealing, and polymerisation temperatures of 94, 55, and 68 C, respectively. After the 30 cycles polymerisation temperature was maintained for 10 min. After cycling the temperature of the PCR products was maintained at 4 C. Purified PCR fragments were cloned using the Directional Topo cloning system into the pET102D/topo vector according to the instructions in the manufacturerÕs manual (Invitrogen). The reaction mixture was used to transform chemically competent Top10 cells (Invitrogen) and recombinant plasmids were isolated using Qiagen purification kits (Qiagen). Clones were analysed by BamH1 restriction sites. Cycle sequencing of positive clones was performed on PCR products amplified with Trxfus (5 0 -TTCCTCGACGCTA ACCTG-3 0 ) and T7ter (5 0 -TAGTTATTGCTCAGCGGT GG-3 0 ) vector specific primers (MilleGen Biotechnologies). Expression and purification of recombinant protein Plasmids containing the N-terminus-repetitive domain of c-Gliadin inserts with the appropriate sequence were transformed into competent BL21(DE3)pLysS cells (Invitrogen) which carries the gene for T7 RNA polymerase under the control of the lacUV5 promoter. Pilot expression Pilot expression experiments were performed using freshly transformed single colonies, grown in 0.05 L cultures of TB medium containing 100 lg/ml ampicilline at 37 C and 250 rpm agitation. At a culture density of A600 = 0.8, the cultures were transferred to incubators at 25, 30 or 37 C. After 20 min of temperature equilibration isopropyl b-D-thiogalactopyranoside was added to a final concentration of 0.5 mM. Aliquots of 1 mL were taken each hour, during 5 h, and the culture density was measured. The cells were harvested at 5000 rpm (bench top centrifuge), during 10 min. The

pellets were resuspended in buffer containing Tris 50 mM, pH 8.0, and 6 M urea, incubated at room temperature for 2 h. The dilution was OD600nm  40 ¼ #lL of urea 6 M; Tris 50 mM added to resuspend the pellet. Cells were lysed by three flash-freeze–thawing cycles. Cell debris was eliminated by centrifugation at 18,000 rpm for 45 min. The supernatants were prepared and electrophoresed on 16% SDS–PAGE gels, with a 6% stacking gel. The optimal temperature and incubation time after induction for a maximal protein expression were determined by the intensity of the band in the SDS gels. Protein medium scale over-expression Single colonies of freshly transformed cells were grown overnight in 4 mL of LB medium in very similar conditions as previously described for pilot expression. The following morning the whole volume of pre-culture was used to inoculate 0.4 L cultures of TB medium containing cultures. In the experiments described in the previous section, it was found that the highest protein expression was achieved at 30 C, and 3 h after IPTG addition. Cells were harvested at 5000 rpm, during 10 min. The pellets were resuspended in buffer containing Tris 50 mM, pH 8.0, and 6 M urea, incubated at room temperature for 1 h. Cells were lysed by ultrasonication during 5 min. Bacterial cell debris was then removed by centrifugation (45 min at 18,000 rpm). The supernatant was filtered (0.20 lm) and loaded to imodacetic acid-resin previously charged with NiSO4 100 mM, and pre-equilibrated with buffer containing 5 M urea, and Tris 50 mM, pH 8.0. To eliminate the non-specific binding proteins, the Ni-column was washed with 50 column volumes of buffer containing 5 M urea, Tris 50 mM, pH 8.0, 400 mM NaCl, and 20 mM Imidazole. The elution of fusion proteins was performed adding three column volumes of buffer containing 5 M urea, Tris 50 mM, pH 8.0, 400 mM NaCl, and 250 mM, Imidazole.

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Cleavage of fusion proteins and identification of the expressed polypeptides

and are expressed as mean residue (DE), the units of which are 103 cm2 mol1.

The fusion proteins were cleaved either by Enterokinase (EK, Biolabs), or by acid cleavage. When using EK, samples were extensively dialysed against Tris 20 mM, NaCl 20 mM, and CaCl2 2 mM, pH 8.0, at room temperature. The digestion was performed following the manufacturer instructions, i.e., 16 h at 23 C, with 6.4 · 103 U of EK per milligram of fusion protein. For performing acid cleavage, the eluted fusion proteins were extensively dialysed against acetic acid 50 mM at room temperature (pH 3.5), afterwards the pH was lowered to a value of 2.0 by the addition of the appropriate volume of 1 M HCl to a final concentration of 10 mM. The digestion was performed at 55 C during 16 h. The EK or acid cleavage of the fusion protein was followed by reversed-phase HPLC (column: Nucleosil C18, ˚ , 4 · 300 mm) using a gradient running from 5 lm, 300 A 100% buffer A (0.1% TFA in H2O) to 100% buffer B (75% acetonitrile, 24.92% H2O, and 0.08% TFA). Samples were analysed using one-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/ PAGE) with 15% gels, with 6% stacking gels and staining with Brilliant Blue R250 (Sigma). Concentrations of purified c-Gliadin solutions were determined from their absorbances at 280 nm, using the absorption coefficient calculated from the amino acid composition [A1cm 0.1% (= 1 g L1) = 0.437 ].

Results Protein expression, purification, and solubility of the fusion protein The N-terminal-repetitive domain of c-Gliadin was over-expressed in fusion with thioredoxin in batch 0.4 L cultures. After 3 h of protein expression the fusion protein appeared in SDS PAGE as a band of about 35 k (Fig. 2). The identity of the protein fusion (TRX-NRG) was confirmed by a Western blot with an antibody specific of the repeated domain of c-Gliadin (not shown). The theoretical MW of the protein fusion is, however, 29,628 (the mature sequence is underlined on Fig. 1). This discrepancy was not surprising because it was found earlier that the repetitive domain of the gliadins was responsible for the overestimation of their MW by SDS–PAGE or gel filtration [50,51]. Partial purification of the fusion protein was carried out, by Ni affinity chromatography. The samples eluted by a buffer containing 5M urea, Tris 50 mM, pH 8.0, 400 mM NaCl, and 250 mM imidazole were extensively dialysed against a buffer Tris 20 mM, NaCl 20 mM, and CaCl2 2 mM, pH 8.0, at room temperature. A visual observation indicated that under these conditions, the samples were mainly soluble and showed only slight aggregation at pH 8.0 or higher at room temperature. Aggregation

Mass spectrometry Mass measurements were performed either on an ion trap mass spectrometer (LCQ Advantage, Thermo-Finnigan, San Jose, USA) or on a quadripole-time-of-flight instrument (Q-TOF Global, Waters/Micromass, Manchester, UK). Both were equipped with an electro-spray ionization source and operated in the positive ion mode. Samples were solubilized at a concentration of ca. 2 nmol mL1 with an aqueous mixture of water and acetonitrile (1:1, vol./vol.) acidified with 0.5% formic acid. Samples were infused into the mass spectrometer at a continuous flow rate of 3 lL min1. Mass data were recorded on the range 500–2000 M/z using the X-Calibur v.1.3 software. Circular dichroism CD measurements were done in a spectropolarimeter (CD6 Jobin Yvon) equipped with a water-jacketed cell holder for temperature control. The concentration of protein was 250 lg mL1 with 0.15% of TFA (pH 1.0). CD spectra were recorded employing 0.5 mm pathlength cells, by averaging 1.0 s at 0.5 nm intervals in the range of 250–197 nm, and 1 nm bandwidth was used. Buffer baselines were subtracted from all spectra. The results presented are an average of at least two measurements

Fig. 2. Expression of fusion protein. SDS–PAGE electrophoresis (16%) stained with Coomassie blue of fusion protein expression. Lane M, shows molecular weight markers; lane C, shows non-induced control, and the third lane shows induced sample after 3 h, incubated at 30 C after induction.

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enhanced at room temperature and pH 3.5 or lower, and aggregation was not detected under these conditions. Cleavage of the fusion protein

was promoted at lower temperatures. To verify whether TRX-NRG fusion protein was mainly soluble or not, the samples that showed evident aggregation at high pH were either mixed by vortexing during 1 min, to get an homogenous suspension or spun down to separate the pellet from the supernatant. Aliquots from suspension, supernatant and pellet were analysed by SDS electrophoresis. As seen on Fig. 3, roughly the same amounts of TRX-NRG were found in the whole suspension (obtained by vortexing) and in the soluble supernatant. On the contrary, the insoluble pellet contained only a low amount of TRX-NRG. This showed that TRX-NRG was mostly soluble even at high pH. The solubility of the fusion protein was notably

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Fig. 3. Solubility of fusion protein. SDS–PAGE electrophoresis (16%) stained with Coomassie blue of partially isolated fusion protein. Lane M, shows molecular weight markers. When incubated at pH 8.0 partially isolated fusion protein showed slight aggregation. To verify whether the fusion protein was mainly soluble or not, the samples were either vortexed to get a suspension of the whole material (W), or spun down to separate supernatant (S) and pellet (P), and electrophoresed.

TRX-NRG consists on a 262-residues-polypeptide (Fig. 1). The first 115 residues correspond to the sequence of His-Patch thioredoxin, followed by the Asp-Asp-AspAsp-Lys specific sequence for enterokinase cleavage. Residues 124 and 125 are Asp-Pro. This pair is quite labile for acid cleavage at high temperatures. Therefore they constitute an acid cleavable site under selected conditions for the recovery of the gliadin repetitive polypeptide [36]. At positions 128–133 a 6-Histidine-tag was introduced by oligonucleotide design, to facilitate the purification by affinity chromatography. The 6-His-tag is immediately flanked by residues Asp-Pro at positions 134 and 135, corresponding, again, to a second acid-cleavable site. The last 127 residues correspond to the sequence of short N-terminal and the repetitive domains of mature c-Gliadin without its signal peptide. It is to note that a third Asp-Pro cleavage sites is located five residues after the N-term of this mature sequence. The presence of one specific site for enterokinase cleavage and three sites potentially labile for acid cleavage (Asp-Pro), allowed us to obtain the N-terminus-repetitive domain of c-Gliadin without its fusion partner by two different ways. After cleavage the polypeptides were separated by reverse-phase chromatography and analysed by mass spectrometry. In the case of enterokinase cleavage, the reverse-phase chromatogram (Fig. 4A) showed two very sharp peaks; the first one eluted around 17.8 % of acetonitrile. This retention time corresponds to that of the repetitive domain isolated from the native c-Gliadin after chymotryptic hydrolysis. This identification was verified by mass spectrometry (Fig. 5A). The experimental mass of the repeated polypeptide was found 16832.0 to compare with the

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Fig. 4. Reverse phase HPLC chromatograms of N-terminal-repetitive domain of c-Gliadin. A lRPC C18, was used with a gradient running from 100% of buffer containing 0.1% TFA in H2O to 100% buffer containing 75% acetonitrile, 24.92% H2O, 0.08% TFA. (A) Protein cleaved by enterokinase. (B) Protein separated by acid cleavage.

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Fig. 5. Electrospray ionisation mass spectra of cleaved N-terminal-repetitive domain of c-Gliadin. (A) Enterokinase-cleaved protein; ‘‘ek’’ stands for enterokinase cleaved NRG. (mass measurements performed on an ion trap instrument). (B) Acid-cleaved protein; ‘‘S1, S2, S3’’ stand for acid cleavage of NRG at site 1, site 2, or site 3, respectively (mass measurements performed on a Q-Tof instrument).

theoretical mass of 16831.43 of the NRG sequence flanked with the 15 amino acid residues of 6-His tag and Asp-Pro cleavage sites. Only one species was detectable. Acid cleavage was carried out at pH 2.1 at 55  C during 16 h to expect a complete reaction. Because there are three acid-labile sites (see Fig. 1) acid cleavage could release three NRG polypeptides with different lengths. Their theoretical molecular mass would be 16432.9, 15149.6 and 14464.8 for cleavages at sites 1, 2, and 3, respectively. It was found that

the protein was mostly cleaved at site 3. The reverse-phase chromatogram of the acid-cleaved sample (Fig. 4B) is very similar to the enterokinase-cleaved sample chromatogram, showing two main peaks, nevertheless, it appears to be less sharp. The identity of samples was also verified by mass spectrometry (Fig. 5B). Although this technique is not quantitative, it is clear that the most abundant species in the first eluting peak is the product of acid cleavage at the third labile site, with a mass of Mr 14465.3 compared

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to the exact mass expected Mr 14464.8, for the protein cleaved at acid-cleavage site 3. The two other peptides correspond to the sites 1 (experimental mass: 16433.2) and 2 (experimental mass: 15150.0). The yield of purification was about 5 mg of purified NRG per litre of culture in non-optimized production conditions. Enterokinase cleavage, is highly specific, but expensive. On the other hand, acid cleavage is easy and cheap to scale-up. In addition, the mass spectrometry analysis of the resulting polypeptides proved that the cleavage reaction reached near-completion in the selected conditions. The presence of a Asp-Pro in position 5 of the N-term sequence of c-Gliadins, resulted, however, in a slightly truncated polypeptide. Prolamins, the main cereal storage proteins, are characterized by their low solubility and their strong aggregation behaviour at neutral pH or higher, but fairly soluble at low pH in organic acid solutions. In some cases, the insolubility is considerable even in the presence of thioredoxin, as for maize c-zein and its N-terminus domain [47]. This is a limitation to the use of enzymatic cleavage. With enterokinase or other neutral and basic proteases, cleavage could be possible only in the case of adequate solubility of the fusion protein at the optimal activity conditions of the enzymes. Thus the acid cleavage could be a most feasible alternative. Circular dichroism Far-UV CD spectra of purified-NRG obtained either by acid or enzymatic cleavages were recorded under the same conditions. No spectral differences were found in the samples, showing that the 6-His tag should not be structured, or at least it does not contribute significantly to the secondary structure of the polypeptide (data not shown). Further circular dichroism experiments were carried out using the A

polypeptide obtained by acid cleavage which contains the whole repetitive domain of c-Gliadin. Fig. 6A shows the far-UV CD spectrum of NRG polypeptide (opened squares) solubilized in 0.15% TFA. NRG polypeptide displayed a Poly(Pro) II conformation, characterized by a broad shoulder around 221 nm and a large negative peak at ca 201 nm. The same structure was observed by Tatham et al. on polypeptides prepared from wheat-endosperm c-Gliadin by chymotrypsin hydrolysis and Reverse phaseHPLC [23]. Fig. 6A (closed triangles) shows the far UV-CD spectrum of such a repetitive gliadin polypeptide (RepGP) recorded in the same conditions as NRG polypeptide. Both spectra showed similar shape. The intensity around the broad shoulder is undistinguishable. In contrast, the intensity of the negative peak is significantly larger for the repetitive gliadin polypeptide than for the recombinant the NRG polypeptide. This shows that the secondary structure of RepGP and NRG polypeptide are similar, but the secondary structure elements, might be slightly larger in RepGP than in NRG, due to small sequence differences as suggested by the mass of RepGP (Mr 14445.9). The stability of the secondary structure of NRG polypeptide was followed by recording far UV-CD spectra at different temperatures after thermal equilibration (Fig. 6B). Two main spectral changes were observed. The intensity around the broad shoulder became more negative. Conversely, the intensity of the large negative peak centred around 201 nm decreased. An isocircular dichroic point was observed at 212 nm. Such behaviour was previously observed with x-Gliadins, the sequence of which is almost entirely composed of Gln-and Pro-rich repeats as NRG polypeptide, suggesting two defined conformations at low and high temperatures [18]. When cooling down the sample, the signal around the shoulder is fully recovered, whereas the intensity of the peak is reversed by about B

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Fig. 6. N-terminal-repetitive domain of c-Gliadin Far-UV CD spectra. Spectra were recorded employing 0.5 mm path-length cells, averaging 1.0 s at 0.5 nm intervals in the range of 250–197 nm, and 1 nm bandwidth was used. Buffer baselines were subtracted from all spectra. (A) Acid cleaved N-terminal-repetitive domain of c-Gliadin after (NRG, open squares) and N-terminal domain polypeptide originated from wheat-endosperm c-Gliadin by chymotrypsine hydrolysis ([23]; RepGP, closed triangles). Both spectra were recorded at the same conditions. (B) Thermal denaturation of NRG was followed by recording far UV-CD spectra at different temperatures after thermal equilibration. Afterwards protein was cooled down up to 20 C to evaluate reversibility of unfolding.

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90%. This indicates that the repetitive domain of the c-Gliadin is susceptible of large but essentially reversible changes of conformation. Such reversibility was previously shown on whole c-Gliadin submitted to high pressures and temperatures [17]. Discussion A few reports concern the expression and characterization of recombinant repetitive domains from prolamins. Ems-McClung and Hainline [47] expressed the N-terminus proline-rich domain of c-zein from maize in fusion with thioredoxin. They reported this fusion protein was four times less soluble than the cysteine-rich C-terminus domain expressed in fusion with thioredoxin. They suggested that the insolubility of full length c-zein results from structural interactions of the N- terminus. The N–terminal domain of a HMW glutenin subunit from wheat was expressed in E. coli [26]. It was found insoluble in water in the absence of SDS. Also, Van Dijk et al. [26] were able to express and characterize the central repetitive domain of high molecular weight (HMW) glutenin subunit. The 16.9 kDa polypeptide obtained by them was readily soluble in 20 mM phosphate and acetate buffers in a wide range of conditions; suggesting that the repetitive domain of HMW proteins is readily soluble in water and that the poor water solubility of the HMW proteins resides in their N and or C terminal domains. Such a soluble central domain was also obtained from a HMW subunit isolated from wheat flour [52]. Repetitive peptides based on the central domains of the HMW glutenin subunits were expressed in E coli, purified and characterized [30]. It was shown that intermolecular interactions and solubility depended on the length of the peptide and the regularity of the repeats [53], the longer and the more regular peptides, being the less water-soluble [54]. In the present work, we have been able to obtain a soluble and stable recombinant repetitive domain of c-Gliadin by producing the polypeptide as a fusion with thioredoxin. The fusion was separated by two means, the specific-site cleavage of enterokinase, and the non-specific, but easy, cheap, and highly efficient acid cleavage, at the Asp-Pro pair. The secondary structure of the N-terminal-repetitive domain of c-Gliadin was characterized by Circular dichroism. The recombinant protein obtained showed structural characteristics and very similar to those reported for the N-terminalrepetitive polypeptide obtained by proteolysis by chymotrypsin of wheat endosperm c-Gliadin and whole x-Gliadins [18,23]. A reversible thermal change of conformation was observed for the recombinant polypeptide. Acknowledgments We thank Professor P.R. Shewry for kindly providing us with the gene of c-Gliadin. CG Benitez-Cardoza

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