Soluble expression and purification of tumor suppressor WT1 and its zinc finger domain

Protein Expression and Purification 85 (2012) 165–172

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Soluble expression and purification of tumor suppressor WT1 and its zinc finger domain Robert D. Fagerlund, Poh Ling Ooi 1, Sigurd M. Wilbanks ⇑ Department of Biochemistry, University of Otago, New Zealand

a r t i c l e

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Article history: Received 15 April 2012 and in revised form 2 August 2012 Available online 10 August 2012 Keywords: WT1 Zinc finger Affinity tags Protein–DNA complex

a b s t r a c t Full length murine WT1 and its zinc finger domain were separately inserted into Escherichia coli expression vectors with various fusion tags on either terminus by Gateway technology (Invitrogen) and expression of soluble protein was assessed. Fusion proteins including the four zinc finger domains of WT1 were used to optimize expression and purification conditions and to characterize WT1:DNA interactions in the absence of WT1:WT1 interactions. Zinc finger protein for in vitro characterization was prepared by IMAC purification of WT1 residues 321–443 with a thioredoxin–hexahistidine N-terminal fusion, followed by 3C protease cleavage to liberate the zinc fingers and cation exchange chromatography to isolate the zinc fingers and reduce the level of the truncated forms. Titration of zinc finger domain with a binding site from the PDGFA promoter gave a Kd of 100 ± 30 nM for the KTS isoform and 130 ± 40 nM for the +KTS isoform. The zinc finger domain was also co-crystallized with a double-stranded DNA oligonucleotide, yielding crystals that diffract to 5.5 Å. Using protocols established for the zinc finger domain, we expressed soluble full-length WT1 with an N-terminal thioredoxin domain and purified the fusion protein by IMAC. In electro-mobility shift assays, purified full-length WT1 bound double-stranded oligonucleotides containing known WT1 binding sites, but not control oligonucleotides. Two molecules of WT1 bind an oligonucleotide presenting the full PDGFA promoter, demonstrating that active full-length WT1 can be produced in E. coli and used to investigate WT1 dimerization in complex with DNA in vitro. Ó 2012 Elsevier Inc. All rights reserved.

Introduction Wilms’ tumor is a pediatric kidney malignancy that effects 1 in 10,000 children under 5 years of age and accounts for 6–8% of all pediatric cancers (for reviews see [1–3]). In more than 50% of Wilms’ tumors the expression level of Wilms’ tumor 1 protein (WT1)2 is reduced and in 10–20% of cases the encoding gene is mutated [4– 8]. WT1 is a transcription factor that controls many growth related genes (reviewed in [9]), including the PLATELET-DERIVED GROWTH FACTOR A-CHAIN (PDGFA) via functional domains in the N-terminus and C-terminus [10] (Supporting information Fig. S1). WT1 has the ⇑ Corresponding author. Address: Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand. E-mail address: [email protected] (S.M. Wilbanks). 1 Present address: Singapore Immunology Network. 2 Abbreviations used: CD, circular dichroism; CBD, chitin binding domain; cv, column volume; DTT, dithiothrietol; EMSA, electrophoretic mobility shift assay; GST, glutathione-S-transferase; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; IMAC, immobilized metal affinity chromatography; IPTG, isopropyl-b-D-thiogalactoside; MS, mass spectroscopy; PDGFA, PLATELET-DERIVED GROWTH FACTOR A-CHAIN gene; Trx, thioredoxin; WT1, Wilms’ tumor 1 protein; zfWT1, zinc finger domain of WT1. 1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.08.002

ability to suppress and enhance transcription through separate domains in the N-terminus [11]; the N-terminus of WT1 also contains sequence that allows self-association [12,13] and interaction with the molecular chaperone Hsp70 [14]. WT1 self-association has been implicated in suppressing transcription as suppression of the PDGFA promoter requires two WT1 sites in the promoter [11,15–17]; however, no specific in vitro experiments have addressed this issue. A reconstituted system to investigate the effect of self-association on DNA binding requires a recombinant source of active, full-length protein. The WT1 C-terminus contains four zinc fingers of Cys2His2 type that bind specific DNA sequences and RNA structural motifs [8,18–21]. There are four major WT1 isoforms that are formed from two independent splicing events of the transcribed mRNA molecule [22]. One splice event includes the 17 amino acids of exon 5 and the other includes residues lysine-threonine-serine (KTS) between zinc fingers 3 and 4. Whereas less is known on the role of the exon 5 splice, the KTS splice has been well documented (reviewed by [23]). The KTS isoform binds DNA with GNG- and TCC-motif sequences to direct WT1 to specific promoter regions [15]. The +KTS isoform has been reported to have a reduced affinity for usual GNG-motif DNA targets and been shown to

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bind RNA and be associated with splicing factors, suggesting the +KTS isoform has a post-transcriptional role [19,22,24,25]. The additional KTS residues occur between zinc fingers 3 and 4 and may interfere with the fourth finger’s specific interactions with DNA [18]. The importance of WT1 for development and disease progression are emphasized in Frasier syndrome and Denys–Drash syndrome, disorders that occur because of improper KTS slicing and premature translational termination of WT1, respectively. Understanding at an atomic level how the zinc fingers interact in a sequence specific manner with DNA is of significant interest. The structure of WT1 zinc finger domain (zfWT1) (KTS) bound to DNA was solved by a combination of X-ray crystallography (using data to 3.15 Å) and NMR techniques, giving a model with the first zinc finger in a novel orientation. Zinc finger 1 is modeled bound into the DNA major groove with the fingertip pointing away from DNA, unlike the canonical orientation of other zinc fingers of WT1 and the archetypal Zif268 zinc fingers that lie almost perpendicular to the proceeding finger and point towards the major groove (see Supplementary information Fig. S2) [26–28]. The first three zinc fingers of WT1 are particularly similar in protein sequence and DNA recognition sequence to those of transcription factor Sp1 (48% sequence identity and conservation of 9 of 12 residues predicted to make direct interactions with DNA, Supplementary information Fig. S3), which binds DNA in the canonical orientation, as judged by NMR chemical shift analysis [29]. Understanding the basis for this discrepancy is relevant as the first finger of WT1 improves the binding affinity to DNA in a base-specific manner [18,26]. It would also be interesting to obtain more structural information on the zfWT1(+KTS) isoform to understand how the insertion of three residues into a loop region can influence nucleic acid binding. Transcriptional control by WT1 may also depend on WT1 selfassociation. Using a reporter gene governed by the PDGFA promoter in transient mammalian cell culture experiments, Wang et al. [11] showed that WT1 suppression of transcription depended on WT1 binding sites either side of the transcriptional start site, whereas deletion of either site caused WT1 to become a transcriptional activator: this result implies WT1 homodimerization may be an important factor for WT1 suppression of the PDGFA promoter. Purified, full-length WT1 would allow in vitro dimerization studies to establish whether WT1 binds DNA as a homodimer. Our objective was to express and purify soluble full length WT1 and the zinc finger domain of both isoforms in Escherichia coli to further characterize WT1 function using in vitro techniques. The zinc finger domain is the most characterized domain of WT1 and has been expressed and purified from E. coli in the native state and by refolding of insoluble protein into a soluble state (see examples [30–33]). In contrast, for in vitro studies recombinant full length WT1 has commonly been expressed in rabbit reticulocyte cell lysates (see examples [12,13,34]). Geng and Carstens [35] previously expressed recombinant WT1 in E. coli; however, no assays were performed to test protein function. We assessed expression of mouse WT1 in E. coli as a cost-effective, straightforward route to protein for in vitro analysis. However, E. coli does not perform the same post-translational modifications that may be important for protein function, nor provide the same variety of chaperones that may be necessary for protein folding and solubility. In particular, a stretch of glycines and prolines might compromise appropriate protein folding in a heterologous host. To optimize WT1 solubility an expression system in E. coli was established using Gateway recombination cloning (Invitrogen) to fuse various affinity tags to either termini of WT1, as the choice of fusion partner can influence solubility of mammalian proteins in E. coli [36,37]. Various E. coli cell lines were evaluated to test if their specific attributes could improve protein solubility.

Methods Bacterial strains and plasmids E. coli strain Rosetta 2 (DE3) (Novagen) was used as the host for expression of WT1 zinc finger domain (zfWT1) and additional strains BL21(DE3) (Novagen), BL21(DE3)-pLysS (Novagen), BL21 Star (DE3) (Invitrogen), BL21 Star (DE3)-pLysS and Origami (Novagen) were used for expression of full length WT1. Strain DB3.1 (Invitrogen) was used for propagation of Gateway vectors (Invitrogen) containing the ccdB gene, and strain DH5a was used for cloning and propagation of all other plasmids. Entry vector pENTR11 and destination vectors pDEST15, pDEST17, and pDEST24 were purchased from Invitrogen. Plasmids pET21d(+) and pET32a(+) from Novagen were converted into Gateway-compatible vectors by insertion of a cassette containing recombination sites and subsequently used as destination vectors. Construction of expression vectors Full length WT1 (+exon 5, KTS) was amplified from mouse cDNA in the plasmid pRc/CMV-WT1(+exon 5, KTS) (courtesy of M.R. Eccles) by PCR using primers WT1-NtermGW-for and WT1NtermGW-rev for N-terminal fusions and WT1-Fatg and WT1-rev for C-terminal fusions (refer to Supplementary information Table S1 for all primer sequences). Length variants for zfWT1 KTS isoform using WT1 (+exon 5, KTS) as a template were inserted into an entry vector appropriate for preparing N-terminal fusion constructs covering residues 321–440 (primers zfWT1NtermGW-for and zfWT1-NtermGW-rev), 321–443 (primers zfWT1-NtermGW-for and Rzf443-Nterm), 321–449 (primers zfWT1-NtermGW-for and WT1-NtermGW-rev), 321–449[Q446 N] (primers zfWT1-NtermGW-for and Rzf449(Q446 N)-Nterm) and 313–449 (primers Fzf313-Nterm and WT1-NtermGW-rev): refer to Supplementary information Fig. S1 for graphical description of length variants. An entry vector for preparing C-terminal fusion constructs was also created that covered residues 321–440 (primers zfWT1-Fatg and zfWT1-rev). An entry vector for producing N-terminal fusion constructs of the zfWT1 +KTS isoform was created using WT1 (+exon 5,+KTS) as a template and included WT1 residues 321–443 (primers zfWT1-NtermGW-for and Rzf443Nterm). PCR products for N-terminal fusions included a 50 3C protease site (sequence to encode amino-acids LEVLFQGP) and a 30 in-frame stop codon and Xho I restriction enzyme site; C-terminal fusions required a 50 ATG start codon. PCR products were digested with Xho I, 50 phosphorylated, and ligated into vector pENTR11 previously digested with Xmn I and Xho I. The WT1 coding regions were transferred from the entry clones to destination vectors by recombination with Clonase (Invitrogen) at 22 °C for 4 h and transformed into DH5a. A conventional construct created with T4 ligase was made of the zfWT1 (KTS) residues 321–443 (amplified with primers zfWT1-NtermGW-for and Rzf443-Nterm) and ligated into pET32a between restriction sites EcoR V and Xho I. Expression of soluble WT1 zinc fingers All zinc finger expression constructs were introduced into Rosetta 2 (DE3) cell line by transformation. For expression trials a culture of 100 mL LB media (including 50 lg/mL ampilcillin and 17 lg/mL chloramphenicol) was inoculated with 1 mL overnight culture and grown by shaking at 37 °C until OD600  0.4. Culture was split with 25 mL placed in each of three 125 mL flasks and each equilibrated at either 18, 28 or 37 °C for 15 min before induction with isopropyl-b-D-thiogalactoside (IPTG) at 0.5 mM final concentration. Samples of 5 mL were pelleted at time points 3 and 6 h

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and overnight and the pellet stored at 20 °C. To lyse cells 0.5 mL HEPES wash buffer (20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)–NaOH, pH 7.5, 0.5 M NaF, 0.1 mM ZnCl2, 12% v/v glycerol, 5 mM b-mercaptoethanol) was added to each pellet and then sonicated. Soluble lysates were clarified by centrifugation at full speed for 1 min. The pellet was resuspended by vortex in 0.5 mL HEPES wash buffer and is presented as the insoluble fraction. For larger amounts of Trx-zfWT1(321–443, KTS), TrxzfWT1(321–443,+KTS) and Trx-zfWT1(321–449, KTS)), cultures were grown in 0.4 L LB medium in 2 L baffled flasks. Cultures were grown until OD600  0.4, transferred to18 °C and incubated for 15 min before induction with 0.5 mM IPTG overnight. Cells were harvested by centrifugation and pellets stored at 20 °C.

Purification of Trx-fused WT1 zinc fingers Batch trials involved 5 mL cell lysates containing His6 tagged proteins bound to 50 lL Co2+ – immobilized metal affinity chromatography (IMAC) resin (Clontech) at 4 °C for 30 min, washed with HEPES wash buffer and eluted with wash buffer supplemented with 500 mM imidazole. Purification was performed from a 0.4 L culture that expressed Trx-zfWT1(321–443) with either KTS isoform. The cell pellet was resuspended in HEPES wash buffer and lysed by sonication. The soluble lysate was passed through 1 ml Zn2+charged HiTrap IMAC column (GE healthcare), washed and eluted with a gradient up to 500 mM imidazole in the same buffer. Protein enriched fractions were pooled and cleaved with 3C protease overnight at 4 °C. Digested protein was centrifuged for 5 min full speed at 4 °C and loaded onto HiTrap SP cation exchange chromatography column (GE Healthcare) equilibrated in 20 mM Bicine, pH 9.0, 0.5 M NaF, 0.1 mM ZnCl2, 12% v/v glycerol, 0.5 mM dithiothrietol (DTT). The zfWT1 was eluted with the same buffer supplemented with 0.5 M NaCl. Fractions enriched in zfWT1 were concentrated with a Vivaspin centrifugal concentrator device (Millipore) and aliquots were stored at 80 °C.

Purification of Trx-fused full-length WT1 A cell pellet from 0.4 L culture that expressed Trx-WT1 was resuspended in HEPES wash buffer, except with 0.5 M NaCl in place of NaF. Cells were lysed by sonication and the soluble lysate was applied to a Zn2+-charged IMAC column and protein was eluted by gradient up to 500 mM imidazole. Individual fractions were aliquoted and stored at -80 °C.

Mass spectroscopy Zinc finger protein for mass spectroscopy analysis was purified from Trx-zfWT1(321–449, KTS) by Zn2+-charged IMAC and cleaved with 3C protease. Protein bands that corresponded to the intact zfWT1and three smaller putative zfWT1 truncations were excised from a Coomassie Brilliant Blue stained SDS–PAGE gel. The excised gel fragments were digested by either trypsin or chymotrypsin and peptides analyzed at the Centre for Protein Research, University of Otago, on a 4800 MALDI tandem time-offlight analyzer (Applied Biosystems). MS and MS/MS default calibration settings were updated on eight calibration spots for each operation mode. All MS spectra were acquired in positive-ion mode with 1,000 laser pulses per sample spot. Data were analyzed using Mascot search software.

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Western blot SDS–PAGE gels were transferred to nitrocellulose membrane by electrophoresis in 20 mM Tris, 150 mM glycine, 20% v/v methanol, 0.05% w/v SDS buffer with criterion blotter (Bio-Rad) at 100 V for 30 min at 4 °C. Primary antibodies used were WT1 C-terminal antibody C-19 (Santa Cruz Bioctechnology) and an anti-His antibody (Qiagen). Enhanced chemiluminesence was used to visualize the western blots. Circular dichroism spectroscopy Circular dichroism (CD) spectra were acquired on an OLIS DCM10 spectrophotometer, using a two-chambered mixing cuvette; one chamber initially contained 0.5 mL zfWT1(KTS) protein at 10 lM and the other chamber contained 0.5 mL of DNA probe Xwt1GCG16 (Supplementary information Table S2) at 10 lM in HEPES wash buffer. CD spectra was taken between 240 nm to 320 nm with readings observed every 2 nm for 30 s. Slit width was 1 nm and temperature held at 20 °C. After collection of initial spectra, the cuvette was inverted several times to allow mixing and incubated at room temperature for 30 min before additional spectra were acquired. CD spectra were repeated in triplicate. Electrophoretic mobility shift assay Electrophoretic mobility shift assays (EMSA) were performed with 32P radio-labeled DNA probes similar to that described by Bardeesy and Pelletier [20]. The PDGFA promoter variants (refer to Supplementary information for construction details and Fig. S4) were amplified by PCR with primers PDGFAmplify-for and PDGFAmplify-rev (Supplementary information Table S1), 400 ng DNA was digested with 10 U of restriction enzyme Xma I at 37 °C overnight, separated by agarose gel electrophoresis and the 200 bp DNA fragment was excised and purified. An amount of 80 ng of purified DNA was radio-labeled at 37 °C for 30 min with Klenow polymerase, 3.3 pmol 32P-a-dCTP and 3.5 pmol dGTP. The DNA was ethanol precipitated, the pellet was rinsed to remove unincorporated nucleotide, and resuspended to 20 nM with 10 mM Tris–HCl (pH 7.5). Short dsDNA probes had a single 50 overhang guanine at both termini and 0.625 pmol of each probe was radio-labeled at 37 °C for 30 min with Klenow polymerase and 2.5 pmol of 32P-a-dCTP, ethanol precipitated to remove unincorporated nucleotide and resuspended to 20 nM with 10 mM Tris–HCl (pH 7.5). Each EMSA condition contained 20 mM HEPES-NaOH (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.1 mM ZnCl2, 0.5 mM DTT, 12% v/v glycerol, 0.02% v/v Triton X-100, 10 lg BSA and 1 lg poly(dI-dC). Reactions with full length WT1 were incubated for 30 min at 25 °C before 10 fmol radio-labeled PDGFA promoter DNA (0.5 lL) was added and incubation continued for 60 min. Reactions with zfWT1 were incubated for 15 min at 25 °C before 10 fmol (0.5 lL) radio-labeled short dsDNA probe was added and incubation continued for 20 min. Reactions were separated on 0.5  TBE (45 mM Tris, 45 mM boric acid, and 1 mM EDTA, adjusted with boric acid to pH 8.3) native polyacrylamide gels (14 cm  16 cm) with either 4% or 6% acrylamide (37.5:1 acrylamide:bisacrylamide) for assays involving full-length WT1 or zfWT1, respectively. Gels were pre-run for 90 min at 200 V in a 4 °C room and cooled with ice-chilled circulated water. Samples were loaded and electrophoresed at 200 V for 60 min for 6% acrylamide gels and 120 min for 4% acrylamide gels, chilled as during the pre-run. Crystallization of WT1 fingers Oligonucleotides for co-crystallization used sequences related to, but not identical to the PDGFA promoter site 1 (Xwt1GCG16,

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Xwt1GCG17) or site 2 (Xwt1TCC16, Xwt1TCC22; Supplementary information Table S2). An equal molar amount of protein (zfWT1(KTS) concentrated to 5.9 mg/mL) and one of four dsDNA probes were mixed to a final concentration of 0.4 mM and incubated at room temperature for 20 min. A Mosquito dispensing robot (TTP LabTech) was used to mix 0.1 lL of protein–DNA with an equal volume of reservoir buffer for hanging drop vapor diffusion crystallization. Crystal trays were incubated at 18 and 4 °C. Crystals were screened on a Rigaku MSC MicroMax 007HF rotating copper anode running at 40 kV and 30 mM with Osmic VariMax optics collimated to 0.3 mm and a Raxis IV++ image plate detector. Results Expression and purification of the WT1 zinc finger domain Initial expression and purification of the WT1 zinc finger domain (zfWT1) was performed to test binding activity to nucleic acids of this domain in isolation of the WT1 N-terminal domains and to establish a system to assist the purification of full-length WT1. The amount of expressed, soluble protein was assessed for a minimal zfWT1 domain (WT1 residues 321–440, KTS isoform; Supplementary information Fig. S1) fused with various affinity tags at either the N-terminus (Trx, glutathione-S-transferase (GST) or His6) or C-terminus (GST or His6) using the Gateway recombination system (Invitrogen). Optimal conditions that yielded the highest ratio of soluble recombinant protein to insoluble recombinant protein were established using induction trials at 18, 28 and 37 °C for either 3, 6 h or overnight. All constructs showed appreciable expression and most were substantially soluble immediately after lysis, with the N-terminal His6 tag considerably less soluble than others (Fig. 1). The condition that expressed the optimal amount of soluble recombinant protein was N-terminal construct TrxzfWT1 induced at 18 °C overnight. The C-terminal fusions displayed modest levels of soluble protein; however, additional experiments revealed His6 fusion construct could not be satisfactorily concentrated after IMAC purification and the GST fusion bound affinity resin with poor affinity (Supplementary information Fig. S5). It was observed the zfWT1 fusion proteins readily precipitated from solution when prepared in phosphate buffered saline (Supplementary information Fig. S6). We improved protein stability by using buffers similar to EMSA binding conditions described by Bardeesy and Pelletier [20] (Supplementary information Fig. S7);

Fig. 1. Expression and solubility of tagged zfWT1. WT1 zinc fingers (residues 321– 440) expressed under optimal conditions with different affinity tags as indicated at top: thioredoxin (Trx), glutathione S-transferase (GST) or hexahistidine (His), on the N- or C-terminus as indicated. The induction conditions were: Trx-zfWT1 at 18 °C overnight, GST-zfWT1 at 28 °C for 6 h, His-zfWT1 at 18 °C overnight, zfWT1-GST at 18 °C for 6 h and zfWT1-His at 18 °C overnight. MWM identifies lane of markers with molecular weights indicated at left. WC refers to whole cell lysate and C refers to clarified lysate. Arrows at right show expected positions of the indicated fusion proteins.

Table 1 Chromatographic behavior of isolated Trx and zfWT1 domains. Chromatography resin

Trx Behavior

zfWT1 behavior

Strong cation exchanger at pH 7.0 or 9.0 Strong anion exchanger at pH 7.0 Heparin affinity, pH 7.0

Did not bind

IMAC HIC – Phenyl group HIC – Octyl group HIC – Butyl group Gel Filtrationa

Bound and eluted Did not bind Bound and eluted Bound and eluted Eluted at 0.63– 0.72 cv

Eluted at high pH and salt Portion bound and eluted Bound but did not elute Bound and eluted Bound and eluted Did not bind Did not bind Eluted a 0.75–0.80 cv

Bound and eluted Did not bind

a Elution volumes are the proportion of 330 mL column volume at which the majority of protein eluted; however, we observed a portion of Trx eluting with zfWT1 (Supplementary information Fig. S9).

generally buffers contained 20 mM HEPES–NaOH (pH 7.5), 0.5 M NaF, 12% v/v glycerol, 0.1 mM ZnCl2 and 0.5 mM DTT. Omission of 0.5 M NaF rendered zfWT1 insoluble above 0.5 mg/mL, in agreement with the finding of Nurmemmedov and Thunnissen [33]. It was shown by gel filtration chromatography that reducing agent was required at time of cell lysis for zfWT1 to elute in a monomeric state rather than as a large soluble aggregate (Supplementary information Fig. S8), presumably by preventing disulfide formation when removed from the reduced cellular environment; 5 mM b-mercaptoethanol was used at lysis during IMAC as either DTT or higher concentrations of b-mercaptoethanol compromised the binding capacity of the IMAC resin. Trx-zfWT1(321–440) fusion protein purified by IMAC was digested with 3C protease and several chromatography techniques were tested to separate the zfWT1 domain from the Trx affinity tag (Table 1; Supplementary information Figs. S8 and S9). Chromatography with strong cation exchange resin was the most effective, which was expected as the predicted isoelectric point for zfWT1 (pI 10.3) is greater and the predicted isoelectric point for Trx (pI 5.4) is less than the column buffer (pH 7.5): cation exchange chromatography was improved with a higher pH and this was used in larger purifications. Hydrophobic interaction chromatography resins had differing affinities for the Trx and zfWT1 so could be used as an alternative separation technique. During IMAC, the zinc finger protein co-eluted with three smaller proteins. Mass spectroscopy (MS) confirmed that full-length zfWT1 was obtained and that the smaller proteins were progressive C-terminal truncations of this domain. MS/MS of tryptic and chymotryptic digests identified peptides with C-terminal residues not expected as cleavage sites for these proteases – therefore representing potential sites of intra-cellular proteolysis – that occurred in the loop regions of the zinc finger motif (Supplementary information Fig. S10). The most C-terminal cleavage site thus identified followed Pro413, which precedes the epitope for the commonly used antibody C-19 (Santa Cruz Biotechnologies). Therefore, it would be expected that the C-19 antibody would not recognize the truncated proteins, and this was confirmed by immunoblot (Fig. 2). To investigate whether proteolytic sensitivity of zfWT1 was specific to the construct lacking the last nine C-terminal residues, which could cause structural instability, proteins that varied in length at both termini were expressed fused to a N-terminal Trx domain. These were constructs: zfWT1(321–443) that has three residues after the minimal zinc finger motif and similar to Zif268 crystal structure [27], construct zfWT1(321–449) that included the full C-terminus, construct zfWT1(321–449[Q446N]) that had altered residue 446 that was predicted to produce a more stable protein (Protparam website – http:// web.expasy.org/protparam/), and construct zfWT1(313–449)

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Fig. 2. Immunoreactivity of WT1 zinc finger truncation products. Trx affinity tag was expressed fused to the N-terminus of WT1 residues (321–440), (321–443), (321–449), (321–449[Q446N]), (313–449) and a control encoded in a non-Gateway vector (non-GW, residues 321–443). Soluble protein (indicated by C above lanes) was purified by IMAC batch purification (E above lanes). Panel (A) shows a Coomassie blue stained SDS–PAGE gel, panel (B) a Western blot probed with a penta-His primary antibody and panel (C) a Western blot probed with a C-19 primary antibody. MWM and filled arrows as in Fig. 1. Unfilled arrows indicate apparent truncation products.

that has an additional eight N-terminal residues (Supplementary information Fig. S1). Expressed proteins were purified by IMAC and fractions analyzed with antibodies penta-His and C-19 (Fig. 2). All constructs yielded four penta-His immuno-reactive proteins, which correspond to intact Trx-zfWT1 fusion protein and smaller truncated versions. A construct that included WT1 residues 321–443 that was made by standard ligation methods (rather than by Gateway recombination) was also truncated. This result shows that variation in length and the additional sequence associated with recombination gene transfer did not appear to affect zinc finger proteolysis. To reduce the contamination with the truncation products a dual tag system using Gateway recombination was created that placed a Trx tag on the N-terminus and either a chitin-binding domain (CBD) or GST tag on the C-terminus of the zinc fingers domain; construct Trx-zfWT1-CBD and Trx-zfWT1-GST, respectively (refer to methods in the Supplementary information for cloning details). Sequential binding by the affinity tags should purify only intact zinc fingers, whereas truncated protein should pass in the flow through. Both constructs induced optimal levels of soluble recombinant protein at 28 °C for 6 h with greatest levels achieved

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with Trx-zfWT1-CBD. Yield of Trx-zfWT1-GST fusion protein purified by IMAC and then GST resin was poor (Supplementary information Fig. S11). In contrast, the Trx-zfWT1-CBD fusion protein was successfully purified first by IMAC and second by chitin resin. Cleavage of the chitin-bound protein liberated the zinc finger domain from both affinity tags and showed the zinc fingers apparently free of truncation contaminant; however, the zinc fingers precipitated following recovery. The dual tag system appeared to have successfully reduced the level of zinc finger contaminants, but failed to produce soluble protein. The single N-terminal Trx tagged zinc fingers domain was purified for further functional experiments with chromatography optimized to limit inclusion of truncated proteins. For the final purification, the zinc finger domain of both isoforms (KTS and +KTS) were purified by IMAC, cleaved with 3C protease, and separated by cation exchange chromatography (Fig. 3). The desired polypeptide was greatly enriched by this process and amenable to in vitro characterization. The dsDNA binding ability of the purified zinc finger domains was investigated by circular dichroism (CD) and EMSA (Fig. 4 and Supplementary information Figs. S12 and S13). Mixing of zfWT1(KTS) with specific DNA probe Xwt1GCG16 caused an increase in ellipticity at 275 nm observed by CD, indicative of unwinding of the DNA helix due to protein binding [28] (Supplementary information Fig. 12). Specific binding of zfWT1 to DNA was shown by EMSA with both zinc finger isoforms, in which both bound the first binding site of the PDGFA promoter oligo (P01a) in a sequence specific manner (Fig. 4). Titration of zinc finger domain gave a Kd of 100 ± 30 nM for the KTS isoform and 130 ± 40 nM for +KTS for the isoform. We also confirmed a report [15] that the KTS zinc finger isoform binds the second binding site of the PDGFA promoter (TCC-motif, probe P02) in a sequence specific manner (Supporting Information Fig. 13). Additionally, we observed the +KTS isoform also binds this site, albeit with 6-fold less affinity than the KTS isoform. Co-crystallization of WT1 zinc fingers with DNA was attempted to characterize these interactions at an atomic level. Crystals grew at 18 and 4 °C overnight only with probe Xwt1GCG16 – a 16-mer dsDNA molecule with 50 single nucleotide overhangs and encoding an optimized WT1 binding sequence related to PDGFA promoter site 1 [18]. The biggest crystals grown at 18 °C were in 35% v/v PEG 3350, 100 mM HEPES–NaOH, pH 7.5 and 0.3 M NaCl, and crystals that grew at 4 °C were in 25% v/v PEG 400 or PEG 350 monomethylethylene, either 0.2 M or 0.45 M NaCl and either 100 mM HEPES–NaOH, pH 7.5 or 100 mM Tris–HCl, pH 8.0 conditions. The crystals formed as hexagonal bipyramids and were approximately 0.1 mM across (Supplementary information Fig. S14). These crystals were tested for X-ray diffraction with home source Rigaku rotating copper anode and diffraction was achieved to 5.5 Å. Further work is required to improve diffraction quality. Expression and purification of full length WT1 Having established an expression and purification system for WT1 zinc fingers, the protocols were applied to full length WT1. The WT1 cDNA was recombined into various expression systems using the Gateway recombination system (Invitrogen). Optimal conditions that expressed the most soluble recombinant protein to insoluble recombinant protein were established using induction trials at 18, 28 and 37 °C for either 3, 6 h or overnight and in various cell lines (summarized in Table 2). The N-terminal fusion protein Trx-WT1 expressed in BL21 Star (DE3)-pLysS cell line displayed the optimal amount of soluble recombinant protein and was purified by Zn2+-charged IMAC with eluted fractions analyzed by SDS–PAGE and visualized by Coomassie blue stain and Western blots with primary antibodies penta-His

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Fig. 3. Purification of zfWT1 for in vitro analysis. Trx-zfWT1(321–443) of both isoforms was purified by IMAC, digested with 3C protease and purified by cation exchange. Panels (A) and (C) show IMAC purification and panels (B) and (D) show cation exchange purification of zfWT1 isoforms (KTS) (panels (A) and (B)) and (+KTS) (panels (C) and (D)). MWM and arrows as in Fig. 2. WC indicates the whole cell lysate, C indicates the soluble clarified lysate, FT indicates the chromatography flow-trough, uncut indicates protein not digested,+3C indicates protein digested with 3C protease and +3C sol indicates the 3C digested sample after centrifugation. The underlined fraction numbers indicate those pooled and retained for further purification or characterization.

Probe

P01a

zfWT1 isoform protein nM

-KTS 0

5

9

19

+KTS

38 75 150 300

5

9

19 38 75 150 300

B

A

Fig. 4. EMSA of WT1 zinc fingers with the first WT1 binding site from the PDGFA promoter (probe P01a). WT1 variant (zfWT1 ± KTS) and concentration used are indicated above each autoradiogram. Label (A) indicates free probe, label (B) indicates the complex of zfWT1 with oligonucleotide.

Table 2 Optimal induction conditions for soluble recombinant WT1 protein. WT1 expressed either with an N-terminal (Trx, GST or His6) or C-terminal (GST, His6) fusion partner. Amount of soluble protein was determined by comparison of protein from 40 ll soluble lysate compared with 500 ng protein standard.

TrxWT1 GSTWT1 HisWT1 WT1GST WT1His

Cell line

Temp. (°C)

Induction time (h)

Soluble proteina

BL21 Star (DE3)pLysS BL21 (DE3)-pLysS

18

6

+++

18

6

++

6

+

BL21 Star (DE3)18 pLysS All tested strains and conditions All tested strains and conditions

 

a Yields indicated by: +++ > 20 ng/l of cell culture, ++ = 20 ng/l of cell culture,+ < 20 ng/l of cell culture (detected only by antibody),  no protein observed.

Fig. 5. Purification by IMAC of full-length fusion of Trx-WT1. Purification was analyzed by SDS–PAGE and Coomassie blue stain or western blot with primary antibodies penta-His and C-19, as identified under each panel. WC refers to whole cell lysate, C refers to clarified lysate and FT refers to the IMAC flow through fraction. MWM and arrows as in Fig. 1.

and C-19 (Fig. 5). The western blots confirmed Trx-WT1 was purified and similar amounts were recovered across eluted fractions. The Trx-WT1 fusion protein was not significantly cleaved by 3C protease (Supplementary information Fig. S15). Furthermore, protein recovered from IMAC bound poorly to cation exchange resin (data not shown); therefore IMAC fractions were not further

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Fig. 6. EMSA of full-length WT1 with PDGFA mutant variants. The radio-labeled probes are as indicated and bound with no or 0.9 lM WT1. Label (A) indicates the free probe, (B) the first band shift representing one WT1 molecule bound, and C the second band shift representing two WT1 molecules bound.

purified. The binding activity of these fractions was investigated with radio-labeled probe synPDGF (sequence that covers the PDGFA promoter and includes two WT1 binding regions). Binding activity varied across the fractions and declined in the later eluting fraction 10 (Supplementary information Fig. S16). EMSAs were performed to test the binding specificity of TrxWT1 protein with radio-labeled versions of the PDGFA promoter that had the two WT1 binding regions specifically disrupted both individually and collectively (Fig. 6). WT1 gave rise to two retarded bands with the intact PDGFA promoter (probes wtPDGF and synPDGF), but to only one band if binding site 2 was mutated (probe mut2) and minimal shifted bands when binding site 1 was mutated, either alone in combination with site 2 (probes mut1 and mut1mut2). Binding of WT1 to site 2 is weak as expected from Wang et al. [15], barely evident with probe mut1 and only obvious by comparison of banding patterns with the synPDGF and mut2 probes. It was observed that Trx alone could not bind the PDGFA promoter, nor could residual native E. coli proteins purified by the protocol used to purify Trx-WT1 (Supplementary information Fig. S17). Together these results support the interpretation that WT1 bound DNA in an expected sequence specific manner and is suitable for further in vitro characterizations to investigate WT1 dimerization. Discussion Soluble full length murine WT1 (+exon 5, KTS) and zfWT1 (+KTS and KTS) have been expressed in E. coli, purified and shown to be functionally active with in vitro DNA binding assays. Each recombinant protein showed increased solubility when fused with a thioredoxin affinity tag and expressed at lower temperatures. N-terminal thioredoxin has previously been observed to be better at aiding soluble recombinant protein expression of other mammalian genes in E. coli compared with GST and His6 [36,37]. This is true of WT1. Full length WT1 fused with either GST or His6 C-terminal tag was insoluble and the zfWT1 fused with GST had poor affinity for GST-resin while zfWT1 fused with His6 could not be concentrated satisfactorily. Furthermore, addition of a C-terminal fusion to zfWT1 with an N-terminal Trx domain compromised zinc finger solubility. These results show WT1 was impaired by these C-terminal tags both during expression and after purification and suggest an N-terminal tag is best for purification of WT1. Additional important factors for WT1 solubility were the buffer conditions. Conditions similar to those used in binding assays by Bardeesy and Pelletier [20] improved solubility. Furthermore, we confirmed 0.5 M NaF was required for concentration of zinc finger protein above 0.5 mg/mL, as first reported by Nurmemmedov and Thunnissen [33]: 0.5 M NaF was not necessary for full length WT1 as expression levels were relatively low compared with zfWT1 and high NaF levels interfered with the EMSA.

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Truncation products were detected and mass spectroscopy determined the likely sites of the proteolytic events were in loops near the C-terminus and were predicted to excise the C-19 antibody epitope from the remaining polypeptide. We suggest that proteolysis of zfWT1 expressed in E. coli may have occurred in previous studies but was not detected because of lack of recognition of products by antibody C-19. The dual tag system with an N-terminal thioredoxin tag and C-terminal chitin-binding domain flanking the zinc finger domains allowed selective purification of full-length products but tag removal did not allow recovery of a soluble product. To obtain zfWT1protein for further analysis we used N-terminal thioredoxin tagged protein; Trx-zfWT1 was purified by IMAC with Zn2+-charged resin, digested with 3C protease to liberate zfWT1 and lastly separated by cation exchange chromatography. The greatest levels of soluble WT1 were expressed in cell line BL21 Star (DE3) carrying plasmid pLysS. This cell line lacks RNase E and reportedly has increased mRNA stability, which may facilitate WT1 expression; the 50 end of the WT1 mRNA has a potential stem-loop secondary structure mediated by a region that encodes a poly-glycine stretch and a complementary sequence downstream that encodes a poly-proline stretch. The Trx-WT1 fusion could not be satisfactorily cleaved and so was assayed as a fusion protein to increase the homogeneity of the final product. Geng and Carstens [35] expressed and purified full length WT1 with fusion partners and also showed heterogeneous WT1 sample containing both cut and full-length polypeptides, although did not present DNA binding information. We present evidence that the activity of WT1 fractions from IMAC had varying activities at binding DNA, particularly a late fraction showed a dramatic drop in activity. Three different experiments confirmed that our purified proteins possess DNA binding ability. First, the ellipticity at 275 nM of a solution of dsDNA increases upon addition of the zfWT1, demonstrating an interaction and consistent with unwinding [28]. Second, extensive EMSA studies support sequence specific binding by both the full-length protein and zfWT1. Titration of protein concentrations used in EMSA against the P01a probe yielded a Kd of the KTS isoform of 100 ± 30 nM, consistent with 140 nM observed for binding to a similar sequence [38]; this suggests that a large fraction of protein purified by our method is active. Isoform +KTS purified in this way gives a much lower Kd (130 ± 40 nM) than published by Drummond (2 lM), suggesting that measures to enhance solubility and minimize truncation can lead to greater homogeneity and specific activity. The full-length construct gave the first in vitro evidence that WT1 may bind some sequences as a functional homodimer. Third, we achieved co-crystals of zfWT1 (KTS) with DNA containing the GCG-motif sequence and designed to twist 1.5 turns per complex. Co-crystals were obtained with the 16-mer containing the GCG-motif sequence, but not with 17-mer probe. If the DNA oligonucleotides create the crystallographic repeat by stacking end-to-end as designed, then there are 32 base pairs to three full turns of the double helix, or 33.75° turn per base-pair. Compared to 36° for ideal B-DNA, this accords with the unwinding we observed by circular dichroism and is similar to the twist of 32.1° per base-pair observed in the crystal structure of the archetypical zinc finger protein Zif268 bound DNA [27,28]. Crystals diffracted to 5.5 Å but a structure has not yet been solved. The only available structure for zfWT1 binding to DNA did not yield a molecular replacement solution with our data, so our crystal may include features not represented in that structure [26]. The oligomeric state of WT1 changes during development suggesting a regulatory role for the dimer [13,39]. Intriguingly, Wang et al. [11] observed WT1 suppressed a reporter gene controlled by the PDGFA promoter, which has WT1 binding sites either side of the transcriptional start site, and deletion of either binding site

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