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Screening of fusion partners for high yield expression and purification of bioactive viscotoxins
Protein Expression and Purification 64 (2009) 16–23
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Protein Expression and Purification j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y p r e p
Screening of fusion partners for high yield expression and purification of bioactive viscotoxins Julius Bogomolovas a, Bernd Simon a, Michael Sattler a,b,c, Gunter Stier a,d,* a
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany c Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany d Umeå Center for Molecular Pathogenes is, Umeå University, SE-901 87 Umeå, Sweden b
a r t i c l e
i n f o
Article history: Received 20 June 2008 and in revised form 2 October 2008 Available online 17 October 2008 Keywords: Viscotoxins Thionins Fusion tag Expression screen Escherichia coli Isotopic labeling Cytotoxicity
a b s t r a c t Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy pos sible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6tagged fusion protein was captured with Ni2+ affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipit ation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 1 l of culture medium corre sponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by het eronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins. © 2008 Elsevier Inc. All rights reserved.
Introduction Viscotoxins are toxic proteins of low molecular weight isolated from European mistletoe (Viscum album L). Based on their origin and the high sequence similarity they belong to a specific class of the plant thionin family. Proteins of this class are found in leaves and stems of mistletoe species. They consist of 45–46 amino acids, are highly basic and share high sequence as well as tertiary structure similarity [1,2]. Three disulfide bridges stabilize the fold, comprising two alpha helices connected by a turn and a short antiparallel beta-sheet. This fold is found in viscotoxin A3 and with minor modifications in other members of III class a/b thionins [3,4]. Thionins are toxic to eukaryotes and prokaryotes in vitro and in vivo suggesting a role in plant defense. Viscotoxins were shown to be toxic to phytophatogenic fungi in vitro [5] and to provide resistance in transgenic plants [6], which implies that viscotoxin * Corresponding author. Address: Umeå Center for Molecular Pathogenesis, Umeå University, SE-901 87 Umeå, Sweden. E-mail addresses: gun[email protected], Gunter.Stier@mpimf-heidelberg. mpg.de (G. Stier). 1046-5928/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.10.003
and – in broader content – thionins function as defense proteins. Nevertheless, in Arabidopsis thaliana the thionin gene is activated by a different pathway than defense proteins [7] casting some doubt on a primary defense function for thionin in A. thaliana. Other reported activities of thionins as chaperones [8] or in DNA binding [9] suggest additional functions of this family of proteins. Thus, the biological role in vivo remains unclear. Thionin cytotox icity is explained by its interaction with the cell membrane, which leads to channel formation, general destabilization or dissolution [10–12], although more complex interactions and effects are also found [13,14]. Despite of high structural similarity the six isoforms of viscotoxins have very different cytotoxic potentials towards cancer cells in vitro [15–17], which may be associated with subtle amino acid substitutions on the solvent accessible surface of the protein leading to different affinities and perturbations of mem branes [18]. Heterologous expression of proteins in Escher ichia coli has been proven to be the most cost-effective and safe method of recombinant protein production, providing the basis for using a variety of methods to understand protein structure and activ ity [19–21]. Problems with proteolytic degradation, low level of
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expression, insolubility and toxicity of thionins and related pro teins can be overcome by expression of the protein of interest as a fusion with carrier proteins [22–27]. The level of solubility and expression of such fusion proteins can be increased by screening induction temperature and E. coli strain [28]. Native viscotoxins, have been used for various studies and are active components in medical mistletoe extract [29–33]. Several atomic structures of thionins have been determined using native sources [3,4,34]. Up to date, only crambin, a non-toxic member of the thionin family was produced by heterologous expression. Fusion of crambin with maltose binding protein in E. coli gave low, but sufficient amounts for structural studies [35]. In other cases heterolog ous expression or in vitro translation was car ried out for non-toxic thionin precursors [36,37], but no quan tification of the expression efficiency was performed. To further enable structural and biophysic al studies, which require larger amounts of highly purified protein, we wished to establish an efficient protocol for the preparation of recombinant viscotox ins. Based on recent developments of expression vectors [38–40], 13 fusion partners for heterolog ous expression of proteins in E. coli were screened for the preparation of recombinant viscotoxin. The induction temperat ure and E. coli strains used were varied and optimized as the most important factors for protein expres sion [28]. All constructs comprised a six-histidine (His6) affinity tag at the N-terminus of the carrier protein and a TEV1 cleavage site for viscotoxin release. Two isoforms (A3 and B), having the highest and the lowest cytotoxicity, respectively, were produced by this method. Cytotoxicity of the purified viscotoxins was tested on HeLa cells. After 15N isotope labeling of viscotoxin in minimal M9 medium, the correct folding of the protein was confirmed by 1 15 H N correlation NMR experiments.
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37 °C overnight while shaking. For the initial expression screen 1 ml of preculture was used to inoculate 9 ml of LB medium sup plemented only with kanamycin and with 2% glucose. For pro duction upscaling, 10 ml of preculture was used to inoculate 1 l of supplemented LB medium. Inocul ates were grown at 37 °C, until OD600 reached 0.6, then cooled down to 30 °C and induced by the addition of IPTG to a final concentration of 0.5 mM. The cells were pelleted after 18 h, frozen in liquid nitrogen and stored at ¡20 °C. For the initial screen 2 ml of culture was spun down, otherwise the pellet from whole volume was used for protein purification. Fusion protein purific ation All subsequent purification procedures were carried out on ice with pre-cooled solutions. Ten milliliters of lysis buffer consisting of buffer A (20 mM Tris, 150 mM NaCl, 10 mM Imidazole, 1 mM Pefab loc (Boehringer-Mannheim), pH 8) supplemented with 0,2% Igepal 40 was used for each gram of pellet. After resuspension, lysozyme (Serva) and DNAse I (Sigma) were added to a final concentration of 25 lg/ml and 5 lg/ml, respectively. The suspension was briefly sonicated and insoluble material was removed by centrifugation at 17,000g for 20 min at 4 °C. The supernatant was applied twice to gravity flow on microspin or Econo columns (Biorad), which were packed with 100 ll for the initial screen or 2 ml for general purifi cation of Ni2+ charged NTA agarose (Qiagen) and equilibrated with lysis buffer. The column was sequentially washed with 10 times the volume of the bead with the following buffers: Buffer A, Buffer A + 1 M NaCl, Buffer A + 25 mM imidazole and eluted with five times the bead volume (or with 150 ll for the initial screen) of Buffer A + 330 mM imidazole. TEV cleavage
Methods Construction of viscotoxin expression vectors The pUC 18 plasmids with cloned precursors of viscotoxin A3 and B was a generous gift from Professor Klaus Apel (Institute of Plant Sciences, Zurich). A pair of primers (sense: 59-GCT ACC ATGG GC AAG AGC TGC TGC CCC ACC ACC-39, antisense: 59-GCT AGG TAC CTT ATT TAG GAT AAT CCG ACG GAC ATG-39) was used to amplify sequences of mature viscotoxin isoforms with NcoI and Acc651 restriction sites suitable for parallel cloning. The cloned and sequenced PCR product was shuttled into 13 different pET system based vectors carrying different N-terminal His6-tagged fusion partners (for detailed information see: http://www.pepcore. embl.de/strains_vectors/vectors/m-series_vectors.html). Plasmids were produced in E. coli DH5 alpha. Successful cloning was ver ified by PCR with passenger and carrier protein specific primers and DNA sequencing. Expression of constructs Unless otherwise indicated the following procedures were applied. The E. coli strain Rossetta (DE3) pLysS (Novagen) was transformed with the set of vectors. The cells where transferred to kanamycin (100 lg/ml) supplemented LB agar plates. Ten mil liliters of LB supplemented with 100 lg/ml kanamycin and 34 lg/ ml chloramphenicol was inoculated. Precultures were grown at
1 Abbreviations used: NMR, nuclear magnetic resonance; TEV, tobacco etch virus; HSQC, heteronuclear single quantum coherence; IPTG, isopropylthiogalactoside; TOCSY, total correlation spectroscopy; MBP, maltose binding protein; GFP, green fluorescent protein; NTA, nitrilotriacetic acid; ESI TOF MS, electrospray ionization time of flight mass spectroscopy.
Affinity purified fusion proteins were concentrated on Centri con Centrifugal Filter with molecular weight cut-off at 15 kD. The buffer of concentrated proteins was exchanged on PD-10 column (Amersham) equilibrated with TEV digest buffer (20 mM Tris pH 8, 150 mM NaCl and 10% glycerol) After the exchange TEV protease was added to a final concentration of 40 lg/ml. Cleavage was per formed for 20 h at room temperature. Viscotoxin purific ation The protein mixture after TEV cleavage was acidified with buffer B (200 mM Na-acetate pH 4, 10% ethanol and 150 mM NaCl). The pre cipitated protein was spun down and the supernatant was loaded on a strong cation exchange column (1 ml SP HiTrap) equilib rated with buffer B on an Akta prime system. Proteins were eluted by a linear gra dient with buffer B + 1 M NaCl. Viscotoxin containing fractions were concentrated on Centricon Centrifugal Filter with a molecul ar weight cut-off at 3 kD. The buffer was exchanged on a Nap 5 column equili brated with a sterile phosphate buffered saline (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl and pH 7.4). The concentration of the fusion protein was evaluated with a commercial Bradford assay (Pierce). For cleaved viscotoxins the concentration was obtained from absorbance at 280 nm with molar extinction coefficient: e = 2980 M¡1cm¡1. Mass spectrometry Viscotoxins were desalted with C4 micro columns and eluted with acetonitrile/water/formic acid (49.5/49.5/1) directly into cap illary nano-flow needles. Spectra were obtained by ESI TOF (elec trospray ionization time of flight) on a Q-Tof 2™ spectrometer. The exact mass was calculated from the resulting spectrum with the deconvolution program MaxEnt1 from Masslynx4.0.
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To produce uniformly 15N-labeled viscotoxin for NMR studies Rossetta (DE3) pLysS cells were grown in a M9 minim al medium supplemented with 15NH4Cl as the sole nitrogen source. Induc tion and purification procedures were performed as described above for the unlabeled peptide. The 15N-labeled viscotoxin eluted from the cation exchange column, was exchanged into 50 mM phosphate buffer, pH 3.6 and 10% D2O was added. Twodimensional 1H 15N HSQC and 3D 1H 15N HSQC–TOCSY NMR spectra on a 0.3 mM sample were acquired at 22 °C on a Bruker DRX600 equipped with cryogenic triple-resonance probes and processed with Topspin. Cytotoxicity assay Cytotoxicity of unlabeled viscotoxins A3 and B on HeLa cells was measured with commercial ATP-based cytotoxicity assay ATP lite™ on EnVision™ plate reader (both Perkin–Elmer) according to manuf acturer protocol. Cells were incubated with ten serial dilu tions of viscotoxins for 72 h before measuring proliferation inhibi tion. The assay was done in triplicate. Results Expression screening
is H x
ST
6
G
ta g B1 G
g
ZZ
ta
Z
M
ar
ke
r
We used 13 different fusion proteins (Supplementary Table 1) for screening of constructs with highest expression levels of visco toxin. All of the expression vectors are updated versions of the
G FP
pETM-series ([21], G. Stier, manuscript in preparation). Expression of soluble fusion protein was observed in all except one construct (Fig. 1): The construct comprising the mature viscotoxin with only an N-terminal His6 tag followed by a TEV protease recognition site was not expressed in this system and was not further analyzed. The overall good correlation between the intensity of the bands of the fusion proteins expressed in the bacteria and those obtained in the clarified lysate and in the purified samples documents the high solubility and proper accessibility of the affinity tag in all constructs. The purity of the fusion proteins after Ni2+ affinity chromatography was evaluated by gel densitometry. Strong faster migrating bands were observed in fusions with small tags (Z tag, ZZ tag and GB1), internal versions of Dsb proteins and monomeric GFP. Tags of higher molecular weight (NusA, MBP) showed low level of co-purified and/or degraded proteins. We expect the expression level to be the limiting factor in our screen since in all cases the amounts of purified proteins did not exceed 10% of the maximal binding capacity of the Ni2+ charged NTA agarose (1.2 mg pro 100 ll according to manufacturer). The amount of purified fusion protein was measured with a commer cial Bradford assay. The theoretical amount of viscotoxin in the expressed fusion was calculated from the mass ratio between viscotoxin and the complete fusion construct. Fig. 2 presents the expression levels of all constructs taking into account the expected amount of viscotoxin in the fusion proteins. Higher expression levels of other fusion constructs were outweighed by the higher proportion of viscotoxin in fusion proteins with smaller carriers. Based on these data, the fusion protein with thi oredoxin gave the best yield of viscotoxin and was chosen for upscaled production.
A M BP D sb A D in sb A ou t D sb C in D sb C Tr ou x t
Isotope labeling and NMR spectroscopy
N us
18
205 116 66 55 44.3 36 29 24 20 17 14.6
kDa
205 116 66 55 44.3 36 29 24 20 17 14.6
kDa
Fig. 1. Initial screening of viscotoxin A3 fusions with different tags as followed on 15% SDS-PAGE. Two milliliters of cell culture were processed as described in the text. Five microliters of the cell lysate (A) or 15 ll of the eluted proteins (B) were loaded per lane. The strong band at approximately 14.6 kD in the whole cell lysate corresponds to lysozyme, which was added to facilitate cell lysis. The thickness of the bands corresponding to the expressed protein in the whole cell lysate correlates very well with the purified protein amount, indicating high solub ility and proper exposition of affinity tag.
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Fig. 2. Expression levels of different fusion proteins and prediction of viscotoxin A3 yields. Whole bar height (black and grey) represents expression level of the con structs evaluated by Bradford assays with the initially screened samples. Black bars show the theoretical amount of viscotoxin in the fusion context.
Upscaled purification The thioredoxin-viscotoxin fusion protein was overexpressed in BL 21 (DE3) cells. After screening vario us temperatures (25, 30 and 37 °C) and induction times (2, 5 and 18 h), we found that an induction for 18 h at 30 °C gives highest expression levels (Supplementary Fig. 1). Cell mass and clarified lysate from this induction protocol are shown in Fig. 3 (Lanes 1 and 2). After Ni2+ affinity chromatography the target protein was highly enriched (Fig. 3; Lane 3). Contaminants with molecul ar weights smaller than 15 kDa were successfully removed during protein concen tration on centrifugal filter devices (data not shown). Overnight incubation with TEV in exchanged buffer gave almost complete release of viscotoxin (Fig. 3; Lane 4). Notably, the standard proce dure for removal of the carrier protein via a Ni2+ affinity column after TEV cleavage was unsuccessful in the viscotoxin purifica tion. Most of the viscotoxin was left bound on the resin together with the cleaved carrier. We did not observe a shift of the peak
205 116 77 55 44.3 36 29 24 20 17
M
1.
2.
3.
4.
5.
6.
19
in analytical size exclusion chromatography before and after TEV cleavage of the thioredoxin-viscotoxin fusion (Supplementary Fig. 3). A small additional peak visible after the TEV cleavage cor responds to impure viscotoxin. These findings suggest that after TEV cleavage most of the viscotoxin tends to associate with the thioredoxin. Nevertheless, we could isolate the viscotoxin by lowering the pH to 4.0 of the cleavage mixture, which resulted in partial precipitation of impurities, cleaved thioredoxin and TEV prote ase (Fig. 3; Lane 6). Insoluble material was spun down and the supernatant was added to a cation exchange column. Two peaks at 15 mS/cm and 25 mS/cm were obtained during gradient elu tion (Fig. 4). The first peak consisted of pure viscotoxin (Fig. 3; Lane 5) and the second mainly of cleaved thioredoxin (Fig. 3; Lane 7). Thirty-one milligrams of thioredoxin-viscotoxin A3 fusion protein was obtained from 1 l of LB culture giving 5.2 mg of pure viscotoxin A3. This corresponds to a 68% recovery of the protein. Similar yields were obtained growing both isoforms of viscotoxin in LB or in M9 medium. The masses of the viscotoxin isoforms determined by ESI TOF MS indicate high purity, and the presence of three disulfide bridges, as well as the absence of any additional modifications or degradations (Fig. 5). Comparing the masses expected for reduced viscotoxins (5151.9 and 5173.9 Da, for A3 and B isoforms, respectively) with the experimentally obtained molecular weights reveals a deficit of 6 Daltons. This most likely reflects the loss of six hydrogen atoms as a result of the formation of three disulfide bonds, as expected for native viscotoxin. Structural integrity We next recorded NMR spectra to confirm the structural integ rity of the recombinant proteins. Comparing the 1H chemical shifts to published results [37] clearly indicates that the native structure of the protein is preserved. The signals in the 1D 1H NMR spec trum (Fig. 6A) are well separated, exhibit narrow line widths, and are characteristic of a well-folded protein. We observe 45 strong amide peaks in the 1H 15N HSQC experiment, which correspond to the expected number of amide groups in the recombinant visco toxin. Out of six expected NH2 signals for asparagine side chains we observed two strong pairs (Fig. 6B; connected by horizontal lines) and one weaker pair that is seen only in a 3D 1H 15N HSQC–TOCSY experiment (data not shown). Thus, the NMR data clearly confirm properly folded viscotoxin A3.
7.
* **
**
14.6 ***
***
Fig. 3. Peptide release and purification of viscotoxin A3-thioredoxin fusion followed by tricine SDS-PAGE (15%). M, protein marker; Lane 1, whole cell lysate; Lane 2 clar ified lysate; Lane 3, fusion after Ni2+ chelate column; Lane 4, TEV cleaved fusion; Lane 5, purified viscotoxin after cation exchange column; Lane 6, precipitate after acidifi cation of the cleavage mixture; Lane 7, second peak from cation exchange column, consisting of cleaved thioredoxin and other impurities. Fusion (*), cleaved thior edoxin (**) and viscotoxin (***) are marked on the gel.
Fig. 4. Cation exchange chromatography profile of the acidified cleavage mixture. Two peaks were obtained during gradient elution, at 15 mS/cm and 25 mS/cm, com prising viscotoxin A3 and cleaved thioredoxin, respectively.
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Cytotoxicity assay Both recombinant viscotoxins exhibited cytotoxic activity towards HeLa cells in micromolar concentrations (Fig. 7). Viscotoxin
B showed 30% proliferation inhibition at 20 lM concentration while the ED50 for recombinant viscotoxin A3 was 3.1 lM when HeLa cells were used. Viscotoxin B showed significantly lower cytotoxicity than A3.
Fig. 5. ESI TOF mass spectrometry of recombinant viscotoxins. Deconvoluted spectra of both isoforms: A3 (A) and B (B) show high purity, no degradation or modification. The deficit of 6 Da in comparison to the theoretical masses expected for reduced viscotoxin indicates the formation of three disulfide bonds (see text).
Fig. 6. 2D 1H lines.
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15
N HSQC spectrum of
21
15
N-labeled viscotoxin A3 in 10% D2O and 90% H2O at pH 3.6 and 22 °C. The side-chain resonances of Asn residues are connected by
Discussion Thionins and related antibacterial peptides have been success fully expressed in E. coli as fusion proteins. The fusion apparently protects these peptides from proteolysis, aggregation and achieves high expression yields [25–27,41,42]. Moreover, the fusion tag often is a prerequisite for allowing the expression of thionins in E. coli by suppressing their antibacterial properties. Similarly, in plants acidic domains found in thionin pre-proteins are believed to have a function in the inhibition of toxicity [37]. Our expres sion screen showed high solubility levels for all protein fusions, indicating that proper folding of viscotoxin in E. coli cells is not a major problem. Thioredoxin has been successfully used as a fusion partner to produce large amounts of soluble thionins or small peptides [23,24,26,41] The fact that thioredoxin enhances expression of disulfide bond containing proteins like defensins and viscotoxins might be explained by temporary reduction of the disulfide bonds that are essential for their structural integrity. However, thiore doxin, where the catalytic cysteines have been mutated, is still
capable to assist the folding of fused antibodies in E. coli cytoplasm [43]. Therefore, an additional/alternative thiol-independent fold ing and enhancing activity of thioredoxin could arise from endog enous chaperones, which tend to associate with thioredoxin [44]. Up to date, the mechanisms of how thioredoxin achieves enhanced expression and/or chaperone activity are poorly understood. We note, that among the 13 different fusion proteins used in this study the thioredoxin fusion proteins were not those that gave the highest overall expression level. However, the thioredoxin fusion was selected since it comprises a high percentage of the target pro tein in the fusion construct, thus providing the highest absolute yield of purified viscotoxin. Our ranking of fusion proteins accord ing to their effect on expression fully matches a recent expression screen of small human proteins in E. coli [45], where a His6 tag alone was found to perform worst and a thioredoxin fusion was found to be best in terms of enhanced expression of the fusion proteins tested. In contrast, our findings are at varia nce with another screen, in which GB1 fusion proteins gave better yields than thioredoxin fusions [46]. Noteworthy, the physicochemic al features of thio nins are rather distinct from typical target proteins used in large
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teins. However, by comparing absolute ED50 values ranging from 0.1 lM to 3 lM obtained in those assays [15–17] recombinant vis cotoxins possessed lover overall toxicity. Due to different assay protocols employed these observations should be taken with caution. The additional GAMG sequence at the N terminus of our recombinant viscotoxins could play role in non-specific attenua tion of the toxicity. The first glycine residue is required for efficient TEV cleavage and the AMG fragment is as a part of Nco I recogni tion site used for viscotoxin cloning (Supplementary Fig. 2). The membrane interacting part of viscotoxins is located in a helical region and the addition of extra N-terminal residues would not be expected to have a specific effect on the membrane interact ing surface. Potentially, it might generate a protruding “tail”, which could reduce specific membrane binding. In conclusion, we provide an efficient expression and purifica tion protocol for the preparation of recombinant viscotoxin in E. coli. This now enables structural and biophysical studies by pro ducing isotope-labeled proteins for binding studies with potential ligands, by employing mutagenes is approaches and for further biochemical characterization of this family of proteins. Fig. 7. Cytotoxicity of recombinant viscotoxins towards HeLa cells. Both isoforms inhibit the proliferation of HeLa cells at micromolar concentrations after incuba tion for 72 h.
recombinant expression and solubility screens [28,40,45,46]. There fore, it may not be surprising that different fusion tags are found to be more suitable for enhancing thionin expression. In this respect, our study is the first comprehensive and systematic expression and solubility screen directed to thionins and related proteins. Different techniques are employed such as ultrafiltration [23], reversed phase [27,41,42], metal-chelate [47], and ion exchange [25] chromatography alone or in combinations to purify the tar get proteins from the cleavage mixture. The standard procedure of carrier removal with Ni2+ affinity column after TEV cleavage was unsuccessful in the viscotoxin purification protocol, as most of the viscotoxin was left bound on resin with the cleaved car rier protein. Association of the thioredoxin with the viscotoxin in cleavage mixture was verified by analytic al size exclusion chro matography (Supplementary Fig. 3). In order to test a potential specific interaction between viscotoxin and thioredoxin NMR titration experiments were performed in PBS buffer. The 1H, 15N HSQC spectrum of thioredoxin alone and after addition of 10-fold excess of unlabeled viscotoxin is virtually unchanged. This argues against a specific interaction between the two proteins. Poten tially, electrostatic interactions between the carrier and passen ger proteins that are persistent after cleavage could mediate an interaction on the column. This is further supported by the oppo site charges of the two proteins at pH 8 in the buffer used for the second Ni2+ affinity column (theoretical pI for viscotoxin A3 and thior edoxin are respectively 9.3 and 5.25). Alternatively, the presence of impurities in the cleavage mixture could facilit ate the observed aggregation. To overcome the interaction between thioredoxin and cleaved viscotoxin an ion exchange chromatog raphy purification was performed, using buffers supplemented with 10% ethan ol at low pH, where both proteins are expected to be positively charged. Ethanol was used instead of glycerol to reduce potential hydrophobic interactions without increasing the viscosity of the solution. In contrast to thioredoxin, TEV prote ase and other impurities that mostly precipitated in acidic con ditions, viscotoxin is stabile in acidic conditions [2,12] and was therefore enriched before the ion exchange chromatography. Thus, a two-step purification procedure was sufficient to obtain pure and well-folded viscotoxins. Recombinant viscotoxin B was significantly less toxic than A3 towards HeLa cells in our assay. This phenomenon has also been observed in several cancer cell line based assays using native pro
Acknowledgments We would like to thank the proteomic core facility at EMBL, in particular Sabrina Rüggeberg for providing mass spectra, Peter Sehr and chemical core facility for cytotoxicity measurement and Gilles Trave for helpful discussions and critical reading of this man uscript. We are grateful to Prof. Klaus Apel for plasmids encoding pre-proteins of viscotoxins. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pep.2008.10.003.
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Protein Expression and Purification j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y p r e p
Screening of fusion partners for high yield expression and purification of bioactive viscotoxins Julius Bogomolovas a, Bernd Simon a, Michael Sattler a,b,c, Gunter Stier a,d,* a
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany c Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany d Umeå Center for Molecular Pathogenes is, Umeå University, SE-901 87 Umeå, Sweden b
a r t i c l e
i n f o
Article history: Received 20 June 2008 and in revised form 2 October 2008 Available online 17 October 2008 Keywords: Viscotoxins Thionins Fusion tag Expression screen Escherichia coli Isotopic labeling Cytotoxicity
a b s t r a c t Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy pos sible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6tagged fusion protein was captured with Ni2+ affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipit ation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2 mg of visctoxin A3 from 1 l of culture medium corre sponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by het eronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins. © 2008 Elsevier Inc. All rights reserved.
Introduction Viscotoxins are toxic proteins of low molecular weight isolated from European mistletoe (Viscum album L). Based on their origin and the high sequence similarity they belong to a specific class of the plant thionin family. Proteins of this class are found in leaves and stems of mistletoe species. They consist of 45–46 amino acids, are highly basic and share high sequence as well as tertiary structure similarity [1,2]. Three disulfide bridges stabilize the fold, comprising two alpha helices connected by a turn and a short antiparallel beta-sheet. This fold is found in viscotoxin A3 and with minor modifications in other members of III class a/b thionins [3,4]. Thionins are toxic to eukaryotes and prokaryotes in vitro and in vivo suggesting a role in plant defense. Viscotoxins were shown to be toxic to phytophatogenic fungi in vitro [5] and to provide resistance in transgenic plants [6], which implies that viscotoxin * Corresponding author. Address: Umeå Center for Molecular Pathogenesis, Umeå University, SE-901 87 Umeå, Sweden. E-mail addresses: gun[email protected], Gunter.Stier@mpimf-heidelberg. mpg.de (G. Stier). 1046-5928/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.10.003
and – in broader content – thionins function as defense proteins. Nevertheless, in Arabidopsis thaliana the thionin gene is activated by a different pathway than defense proteins [7] casting some doubt on a primary defense function for thionin in A. thaliana. Other reported activities of thionins as chaperones [8] or in DNA binding [9] suggest additional functions of this family of proteins. Thus, the biological role in vivo remains unclear. Thionin cytotox icity is explained by its interaction with the cell membrane, which leads to channel formation, general destabilization or dissolution [10–12], although more complex interactions and effects are also found [13,14]. Despite of high structural similarity the six isoforms of viscotoxins have very different cytotoxic potentials towards cancer cells in vitro [15–17], which may be associated with subtle amino acid substitutions on the solvent accessible surface of the protein leading to different affinities and perturbations of mem branes [18]. Heterologous expression of proteins in Escher ichia coli has been proven to be the most cost-effective and safe method of recombinant protein production, providing the basis for using a variety of methods to understand protein structure and activ ity [19–21]. Problems with proteolytic degradation, low level of
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expression, insolubility and toxicity of thionins and related pro teins can be overcome by expression of the protein of interest as a fusion with carrier proteins [22–27]. The level of solubility and expression of such fusion proteins can be increased by screening induction temperature and E. coli strain [28]. Native viscotoxins, have been used for various studies and are active components in medical mistletoe extract [29–33]. Several atomic structures of thionins have been determined using native sources [3,4,34]. Up to date, only crambin, a non-toxic member of the thionin family was produced by heterologous expression. Fusion of crambin with maltose binding protein in E. coli gave low, but sufficient amounts for structural studies [35]. In other cases heterolog ous expression or in vitro translation was car ried out for non-toxic thionin precursors [36,37], but no quan tification of the expression efficiency was performed. To further enable structural and biophysic al studies, which require larger amounts of highly purified protein, we wished to establish an efficient protocol for the preparation of recombinant viscotox ins. Based on recent developments of expression vectors [38–40], 13 fusion partners for heterolog ous expression of proteins in E. coli were screened for the preparation of recombinant viscotoxin. The induction temperat ure and E. coli strains used were varied and optimized as the most important factors for protein expres sion [28]. All constructs comprised a six-histidine (His6) affinity tag at the N-terminus of the carrier protein and a TEV1 cleavage site for viscotoxin release. Two isoforms (A3 and B), having the highest and the lowest cytotoxicity, respectively, were produced by this method. Cytotoxicity of the purified viscotoxins was tested on HeLa cells. After 15N isotope labeling of viscotoxin in minimal M9 medium, the correct folding of the protein was confirmed by 1 15 H N correlation NMR experiments.
17
37 °C overnight while shaking. For the initial expression screen 1 ml of preculture was used to inoculate 9 ml of LB medium sup plemented only with kanamycin and with 2% glucose. For pro duction upscaling, 10 ml of preculture was used to inoculate 1 l of supplemented LB medium. Inocul ates were grown at 37 °C, until OD600 reached 0.6, then cooled down to 30 °C and induced by the addition of IPTG to a final concentration of 0.5 mM. The cells were pelleted after 18 h, frozen in liquid nitrogen and stored at ¡20 °C. For the initial screen 2 ml of culture was spun down, otherwise the pellet from whole volume was used for protein purification. Fusion protein purific ation All subsequent purification procedures were carried out on ice with pre-cooled solutions. Ten milliliters of lysis buffer consisting of buffer A (20 mM Tris, 150 mM NaCl, 10 mM Imidazole, 1 mM Pefab loc (Boehringer-Mannheim), pH 8) supplemented with 0,2% Igepal 40 was used for each gram of pellet. After resuspension, lysozyme (Serva) and DNAse I (Sigma) were added to a final concentration of 25 lg/ml and 5 lg/ml, respectively. The suspension was briefly sonicated and insoluble material was removed by centrifugation at 17,000g for 20 min at 4 °C. The supernatant was applied twice to gravity flow on microspin or Econo columns (Biorad), which were packed with 100 ll for the initial screen or 2 ml for general purifi cation of Ni2+ charged NTA agarose (Qiagen) and equilibrated with lysis buffer. The column was sequentially washed with 10 times the volume of the bead with the following buffers: Buffer A, Buffer A + 1 M NaCl, Buffer A + 25 mM imidazole and eluted with five times the bead volume (or with 150 ll for the initial screen) of Buffer A + 330 mM imidazole. TEV cleavage
Methods Construction of viscotoxin expression vectors The pUC 18 plasmids with cloned precursors of viscotoxin A3 and B was a generous gift from Professor Klaus Apel (Institute of Plant Sciences, Zurich). A pair of primers (sense: 59-GCT ACC ATGG GC AAG AGC TGC TGC CCC ACC ACC-39, antisense: 59-GCT AGG TAC CTT ATT TAG GAT AAT CCG ACG GAC ATG-39) was used to amplify sequences of mature viscotoxin isoforms with NcoI and Acc651 restriction sites suitable for parallel cloning. The cloned and sequenced PCR product was shuttled into 13 different pET system based vectors carrying different N-terminal His6-tagged fusion partners (for detailed information see: http://www.pepcore. embl.de/strains_vectors/vectors/m-series_vectors.html). Plasmids were produced in E. coli DH5 alpha. Successful cloning was ver ified by PCR with passenger and carrier protein specific primers and DNA sequencing. Expression of constructs Unless otherwise indicated the following procedures were applied. The E. coli strain Rossetta (DE3) pLysS (Novagen) was transformed with the set of vectors. The cells where transferred to kanamycin (100 lg/ml) supplemented LB agar plates. Ten mil liliters of LB supplemented with 100 lg/ml kanamycin and 34 lg/ ml chloramphenicol was inoculated. Precultures were grown at
1 Abbreviations used: NMR, nuclear magnetic resonance; TEV, tobacco etch virus; HSQC, heteronuclear single quantum coherence; IPTG, isopropylthiogalactoside; TOCSY, total correlation spectroscopy; MBP, maltose binding protein; GFP, green fluorescent protein; NTA, nitrilotriacetic acid; ESI TOF MS, electrospray ionization time of flight mass spectroscopy.
Affinity purified fusion proteins were concentrated on Centri con Centrifugal Filter with molecular weight cut-off at 15 kD. The buffer of concentrated proteins was exchanged on PD-10 column (Amersham) equilibrated with TEV digest buffer (20 mM Tris pH 8, 150 mM NaCl and 10% glycerol) After the exchange TEV protease was added to a final concentration of 40 lg/ml. Cleavage was per formed for 20 h at room temperature. Viscotoxin purific ation The protein mixture after TEV cleavage was acidified with buffer B (200 mM Na-acetate pH 4, 10% ethanol and 150 mM NaCl). The pre cipitated protein was spun down and the supernatant was loaded on a strong cation exchange column (1 ml SP HiTrap) equilib rated with buffer B on an Akta prime system. Proteins were eluted by a linear gra dient with buffer B + 1 M NaCl. Viscotoxin containing fractions were concentrated on Centricon Centrifugal Filter with a molecul ar weight cut-off at 3 kD. The buffer was exchanged on a Nap 5 column equili brated with a sterile phosphate buffered saline (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl and pH 7.4). The concentration of the fusion protein was evaluated with a commercial Bradford assay (Pierce). For cleaved viscotoxins the concentration was obtained from absorbance at 280 nm with molar extinction coefficient: e = 2980 M¡1cm¡1. Mass spectrometry Viscotoxins were desalted with C4 micro columns and eluted with acetonitrile/water/formic acid (49.5/49.5/1) directly into cap illary nano-flow needles. Spectra were obtained by ESI TOF (elec trospray ionization time of flight) on a Q-Tof 2™ spectrometer. The exact mass was calculated from the resulting spectrum with the deconvolution program MaxEnt1 from Masslynx4.0.
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To produce uniformly 15N-labeled viscotoxin for NMR studies Rossetta (DE3) pLysS cells were grown in a M9 minim al medium supplemented with 15NH4Cl as the sole nitrogen source. Induc tion and purification procedures were performed as described above for the unlabeled peptide. The 15N-labeled viscotoxin eluted from the cation exchange column, was exchanged into 50 mM phosphate buffer, pH 3.6 and 10% D2O was added. Twodimensional 1H 15N HSQC and 3D 1H 15N HSQC–TOCSY NMR spectra on a 0.3 mM sample were acquired at 22 °C on a Bruker DRX600 equipped with cryogenic triple-resonance probes and processed with Topspin. Cytotoxicity assay Cytotoxicity of unlabeled viscotoxins A3 and B on HeLa cells was measured with commercial ATP-based cytotoxicity assay ATP lite™ on EnVision™ plate reader (both Perkin–Elmer) according to manuf acturer protocol. Cells were incubated with ten serial dilu tions of viscotoxins for 72 h before measuring proliferation inhibi tion. The assay was done in triplicate. Results Expression screening
is H x
ST
6
G
ta g B1 G
g
ZZ
ta
Z
M
ar
ke
r
We used 13 different fusion proteins (Supplementary Table 1) for screening of constructs with highest expression levels of visco toxin. All of the expression vectors are updated versions of the
G FP
pETM-series ([21], G. Stier, manuscript in preparation). Expression of soluble fusion protein was observed in all except one construct (Fig. 1): The construct comprising the mature viscotoxin with only an N-terminal His6 tag followed by a TEV protease recognition site was not expressed in this system and was not further analyzed. The overall good correlation between the intensity of the bands of the fusion proteins expressed in the bacteria and those obtained in the clarified lysate and in the purified samples documents the high solubility and proper accessibility of the affinity tag in all constructs. The purity of the fusion proteins after Ni2+ affinity chromatography was evaluated by gel densitometry. Strong faster migrating bands were observed in fusions with small tags (Z tag, ZZ tag and GB1), internal versions of Dsb proteins and monomeric GFP. Tags of higher molecular weight (NusA, MBP) showed low level of co-purified and/or degraded proteins. We expect the expression level to be the limiting factor in our screen since in all cases the amounts of purified proteins did not exceed 10% of the maximal binding capacity of the Ni2+ charged NTA agarose (1.2 mg pro 100 ll according to manufacturer). The amount of purified fusion protein was measured with a commer cial Bradford assay. The theoretical amount of viscotoxin in the expressed fusion was calculated from the mass ratio between viscotoxin and the complete fusion construct. Fig. 2 presents the expression levels of all constructs taking into account the expected amount of viscotoxin in the fusion proteins. Higher expression levels of other fusion constructs were outweighed by the higher proportion of viscotoxin in fusion proteins with smaller carriers. Based on these data, the fusion protein with thi oredoxin gave the best yield of viscotoxin and was chosen for upscaled production.
A M BP D sb A D in sb A ou t D sb C in D sb C Tr ou x t
Isotope labeling and NMR spectroscopy
N us
18
205 116 66 55 44.3 36 29 24 20 17 14.6
kDa
205 116 66 55 44.3 36 29 24 20 17 14.6
kDa
Fig. 1. Initial screening of viscotoxin A3 fusions with different tags as followed on 15% SDS-PAGE. Two milliliters of cell culture were processed as described in the text. Five microliters of the cell lysate (A) or 15 ll of the eluted proteins (B) were loaded per lane. The strong band at approximately 14.6 kD in the whole cell lysate corresponds to lysozyme, which was added to facilitate cell lysis. The thickness of the bands corresponding to the expressed protein in the whole cell lysate correlates very well with the purified protein amount, indicating high solub ility and proper exposition of affinity tag.
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Fig. 2. Expression levels of different fusion proteins and prediction of viscotoxin A3 yields. Whole bar height (black and grey) represents expression level of the con structs evaluated by Bradford assays with the initially screened samples. Black bars show the theoretical amount of viscotoxin in the fusion context.
Upscaled purification The thioredoxin-viscotoxin fusion protein was overexpressed in BL 21 (DE3) cells. After screening vario us temperatures (25, 30 and 37 °C) and induction times (2, 5 and 18 h), we found that an induction for 18 h at 30 °C gives highest expression levels (Supplementary Fig. 1). Cell mass and clarified lysate from this induction protocol are shown in Fig. 3 (Lanes 1 and 2). After Ni2+ affinity chromatography the target protein was highly enriched (Fig. 3; Lane 3). Contaminants with molecul ar weights smaller than 15 kDa were successfully removed during protein concen tration on centrifugal filter devices (data not shown). Overnight incubation with TEV in exchanged buffer gave almost complete release of viscotoxin (Fig. 3; Lane 4). Notably, the standard proce dure for removal of the carrier protein via a Ni2+ affinity column after TEV cleavage was unsuccessful in the viscotoxin purifica tion. Most of the viscotoxin was left bound on the resin together with the cleaved carrier. We did not observe a shift of the peak
205 116 77 55 44.3 36 29 24 20 17
M
1.
2.
3.
4.
5.
6.
19
in analytical size exclusion chromatography before and after TEV cleavage of the thioredoxin-viscotoxin fusion (Supplementary Fig. 3). A small additional peak visible after the TEV cleavage cor responds to impure viscotoxin. These findings suggest that after TEV cleavage most of the viscotoxin tends to associate with the thioredoxin. Nevertheless, we could isolate the viscotoxin by lowering the pH to 4.0 of the cleavage mixture, which resulted in partial precipitation of impurities, cleaved thioredoxin and TEV prote ase (Fig. 3; Lane 6). Insoluble material was spun down and the supernatant was added to a cation exchange column. Two peaks at 15 mS/cm and 25 mS/cm were obtained during gradient elu tion (Fig. 4). The first peak consisted of pure viscotoxin (Fig. 3; Lane 5) and the second mainly of cleaved thioredoxin (Fig. 3; Lane 7). Thirty-one milligrams of thioredoxin-viscotoxin A3 fusion protein was obtained from 1 l of LB culture giving 5.2 mg of pure viscotoxin A3. This corresponds to a 68% recovery of the protein. Similar yields were obtained growing both isoforms of viscotoxin in LB or in M9 medium. The masses of the viscotoxin isoforms determined by ESI TOF MS indicate high purity, and the presence of three disulfide bridges, as well as the absence of any additional modifications or degradations (Fig. 5). Comparing the masses expected for reduced viscotoxins (5151.9 and 5173.9 Da, for A3 and B isoforms, respectively) with the experimentally obtained molecular weights reveals a deficit of 6 Daltons. This most likely reflects the loss of six hydrogen atoms as a result of the formation of three disulfide bonds, as expected for native viscotoxin. Structural integrity We next recorded NMR spectra to confirm the structural integ rity of the recombinant proteins. Comparing the 1H chemical shifts to published results [37] clearly indicates that the native structure of the protein is preserved. The signals in the 1D 1H NMR spec trum (Fig. 6A) are well separated, exhibit narrow line widths, and are characteristic of a well-folded protein. We observe 45 strong amide peaks in the 1H 15N HSQC experiment, which correspond to the expected number of amide groups in the recombinant visco toxin. Out of six expected NH2 signals for asparagine side chains we observed two strong pairs (Fig. 6B; connected by horizontal lines) and one weaker pair that is seen only in a 3D 1H 15N HSQC–TOCSY experiment (data not shown). Thus, the NMR data clearly confirm properly folded viscotoxin A3.
7.
* **
**
14.6 ***
***
Fig. 3. Peptide release and purification of viscotoxin A3-thioredoxin fusion followed by tricine SDS-PAGE (15%). M, protein marker; Lane 1, whole cell lysate; Lane 2 clar ified lysate; Lane 3, fusion after Ni2+ chelate column; Lane 4, TEV cleaved fusion; Lane 5, purified viscotoxin after cation exchange column; Lane 6, precipitate after acidifi cation of the cleavage mixture; Lane 7, second peak from cation exchange column, consisting of cleaved thioredoxin and other impurities. Fusion (*), cleaved thior edoxin (**) and viscotoxin (***) are marked on the gel.
Fig. 4. Cation exchange chromatography profile of the acidified cleavage mixture. Two peaks were obtained during gradient elution, at 15 mS/cm and 25 mS/cm, com prising viscotoxin A3 and cleaved thioredoxin, respectively.
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Cytotoxicity assay Both recombinant viscotoxins exhibited cytotoxic activity towards HeLa cells in micromolar concentrations (Fig. 7). Viscotoxin
B showed 30% proliferation inhibition at 20 lM concentration while the ED50 for recombinant viscotoxin A3 was 3.1 lM when HeLa cells were used. Viscotoxin B showed significantly lower cytotoxicity than A3.
Fig. 5. ESI TOF mass spectrometry of recombinant viscotoxins. Deconvoluted spectra of both isoforms: A3 (A) and B (B) show high purity, no degradation or modification. The deficit of 6 Da in comparison to the theoretical masses expected for reduced viscotoxin indicates the formation of three disulfide bonds (see text).
Fig. 6. 2D 1H lines.
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15
N HSQC spectrum of
21
15
N-labeled viscotoxin A3 in 10% D2O and 90% H2O at pH 3.6 and 22 °C. The side-chain resonances of Asn residues are connected by
Discussion Thionins and related antibacterial peptides have been success fully expressed in E. coli as fusion proteins. The fusion apparently protects these peptides from proteolysis, aggregation and achieves high expression yields [25–27,41,42]. Moreover, the fusion tag often is a prerequisite for allowing the expression of thionins in E. coli by suppressing their antibacterial properties. Similarly, in plants acidic domains found in thionin pre-proteins are believed to have a function in the inhibition of toxicity [37]. Our expres sion screen showed high solubility levels for all protein fusions, indicating that proper folding of viscotoxin in E. coli cells is not a major problem. Thioredoxin has been successfully used as a fusion partner to produce large amounts of soluble thionins or small peptides [23,24,26,41] The fact that thioredoxin enhances expression of disulfide bond containing proteins like defensins and viscotoxins might be explained by temporary reduction of the disulfide bonds that are essential for their structural integrity. However, thiore doxin, where the catalytic cysteines have been mutated, is still
capable to assist the folding of fused antibodies in E. coli cytoplasm [43]. Therefore, an additional/alternative thiol-independent fold ing and enhancing activity of thioredoxin could arise from endog enous chaperones, which tend to associate with thioredoxin [44]. Up to date, the mechanisms of how thioredoxin achieves enhanced expression and/or chaperone activity are poorly understood. We note, that among the 13 different fusion proteins used in this study the thioredoxin fusion proteins were not those that gave the highest overall expression level. However, the thioredoxin fusion was selected since it comprises a high percentage of the target pro tein in the fusion construct, thus providing the highest absolute yield of purified viscotoxin. Our ranking of fusion proteins accord ing to their effect on expression fully matches a recent expression screen of small human proteins in E. coli [45], where a His6 tag alone was found to perform worst and a thioredoxin fusion was found to be best in terms of enhanced expression of the fusion proteins tested. In contrast, our findings are at varia nce with another screen, in which GB1 fusion proteins gave better yields than thioredoxin fusions [46]. Noteworthy, the physicochemic al features of thio nins are rather distinct from typical target proteins used in large
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teins. However, by comparing absolute ED50 values ranging from 0.1 lM to 3 lM obtained in those assays [15–17] recombinant vis cotoxins possessed lover overall toxicity. Due to different assay protocols employed these observations should be taken with caution. The additional GAMG sequence at the N terminus of our recombinant viscotoxins could play role in non-specific attenua tion of the toxicity. The first glycine residue is required for efficient TEV cleavage and the AMG fragment is as a part of Nco I recogni tion site used for viscotoxin cloning (Supplementary Fig. 2). The membrane interacting part of viscotoxins is located in a helical region and the addition of extra N-terminal residues would not be expected to have a specific effect on the membrane interact ing surface. Potentially, it might generate a protruding “tail”, which could reduce specific membrane binding. In conclusion, we provide an efficient expression and purifica tion protocol for the preparation of recombinant viscotoxin in E. coli. This now enables structural and biophysical studies by pro ducing isotope-labeled proteins for binding studies with potential ligands, by employing mutagenes is approaches and for further biochemical characterization of this family of proteins. Fig. 7. Cytotoxicity of recombinant viscotoxins towards HeLa cells. Both isoforms inhibit the proliferation of HeLa cells at micromolar concentrations after incuba tion for 72 h.
recombinant expression and solubility screens [28,40,45,46]. There fore, it may not be surprising that different fusion tags are found to be more suitable for enhancing thionin expression. In this respect, our study is the first comprehensive and systematic expression and solubility screen directed to thionins and related proteins. Different techniques are employed such as ultrafiltration [23], reversed phase [27,41,42], metal-chelate [47], and ion exchange [25] chromatography alone or in combinations to purify the tar get proteins from the cleavage mixture. The standard procedure of carrier removal with Ni2+ affinity column after TEV cleavage was unsuccessful in the viscotoxin purification protocol, as most of the viscotoxin was left bound on resin with the cleaved car rier protein. Association of the thioredoxin with the viscotoxin in cleavage mixture was verified by analytic al size exclusion chro matography (Supplementary Fig. 3). In order to test a potential specific interaction between viscotoxin and thioredoxin NMR titration experiments were performed in PBS buffer. The 1H, 15N HSQC spectrum of thioredoxin alone and after addition of 10-fold excess of unlabeled viscotoxin is virtually unchanged. This argues against a specific interaction between the two proteins. Poten tially, electrostatic interactions between the carrier and passen ger proteins that are persistent after cleavage could mediate an interaction on the column. This is further supported by the oppo site charges of the two proteins at pH 8 in the buffer used for the second Ni2+ affinity column (theoretical pI for viscotoxin A3 and thior edoxin are respectively 9.3 and 5.25). Alternatively, the presence of impurities in the cleavage mixture could facilit ate the observed aggregation. To overcome the interaction between thioredoxin and cleaved viscotoxin an ion exchange chromatog raphy purification was performed, using buffers supplemented with 10% ethan ol at low pH, where both proteins are expected to be positively charged. Ethanol was used instead of glycerol to reduce potential hydrophobic interactions without increasing the viscosity of the solution. In contrast to thioredoxin, TEV prote ase and other impurities that mostly precipitated in acidic con ditions, viscotoxin is stabile in acidic conditions [2,12] and was therefore enriched before the ion exchange chromatography. Thus, a two-step purification procedure was sufficient to obtain pure and well-folded viscotoxins. Recombinant viscotoxin B was significantly less toxic than A3 towards HeLa cells in our assay. This phenomenon has also been observed in several cancer cell line based assays using native pro
Acknowledgments We would like to thank the proteomic core facility at EMBL, in particular Sabrina Rüggeberg for providing mass spectra, Peter Sehr and chemical core facility for cytotoxicity measurement and Gilles Trave for helpful discussions and critical reading of this man uscript. We are grateful to Prof. Klaus Apel for plasmids encoding pre-proteins of viscotoxins. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pep.2008.10.003.
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