Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1

Protein Expression and PuriWcation 46 (2006) 379–389 www.elsevier.com/locate/yprep

Expression, puriWcation, and characterization of the 4 zinc Wnger region of human tumor suppressor WT1 Elmar Nurmemmedov, Marjolein Thunnissen ¤ Department of Molecular Biophysics, Lund University, Chemical Center, Box 124, SE 221 00, Lund, Sweden Received 11 July 2005, and in revised form 26 October 2005 Available online 22 November 2005

Abstract Wilm’s Tumor gene 1 (WT1) encodes a zinc Wnger protein with four distinct splice isoforms. WT1 has a critical role in genesis of various cancer types both at the DNA/RNA and the protein level. The zinc-Wnger DNA-binding capacity of the protein is located in the C-terminal domain. Two recombinant proteins, 6HIS-ZN¡wt1 and 6HIS-ZN+wt1, corresponding to two alternative splice variants of the C-terminal regions of human WT1 (¡KTS) and WT1 (+KTS), respectively, were over-expressed with hexa-histidine fusion tags in inclusion bodies in Escherichia coli for crystallization studies. A combination of Ni2+–NTA aYnity and size-exclusion chromatography was applied for puriWcation of the proteins in denaturing conditions. The eVects of various buVers, salts and other additives were scrutinized in a systematic screening to establish the optimal conditions for solubility and refolding of the recombinant WT1 proteins. Circular dichroism analysis revealed the expected  content for the refolded proteins, with a notable degradation of the -helical segment in the DNA-free state. Electrophoretic mobility shift assay with double-stranded DNA containing the double Egr1 consensus site 5⬘-GCG-T GG-GCG-3⬘ conWrmed that 6HIS-ZN¡wt1 has higher DNA binding aYnity than 6HIS-ZN+wt1. © 2005 Elsevier Inc. All rights reserved. Keywords: WT1; Zinc Wnger; PuriWcation; Refolding; Secondary structure; Protein–DNA complex

Wilm’s Tumor gene 1 protein (WT1)1 is a zinc Wnger transcription factor with 4 Cys2-His2-type zinc Wngers. The biology of WT1 is complex and it has been shown that the protein, in addition to its role as a tumour suppressor, has multiple roles that are critical for development and maintenance of body function [1,2]. WT1 is expressed during mammalian embryonic development in many tissues, including the urogenital system, spleen, certain areas of the brain, spinal cord, mesothelial organs, diaphragm, limb, and more [3]. There is a variety of organs such as kidneys, gonads and other mesodermally derived organs for which

*

Corresponding author. Fax: +46 46 222 46 92. E-mail addresses: [email protected] (E. Nurmemmedov), [email protected] (M. Thunnissen). 1 Abbreviations used: WT1, Wilm’s Tumor gene 1; DTT, dithiothreitol; GSH, glutathione; GSSH, oxidized glutathione; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; HRP, horseradish peroxidase. 1046-5928/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.10.029

WT1 during development is essential. Mice, in which the WT1 gene is disrupted, die at mid-gestation and this appears to be due to cardiac defects caused by mesothelial abnormalities. These mice also lack adrenal glands and spleen [4]. There are also indications that WT1 plays a role in neuronal development [5]. The common theme for WT1 function during the development of all these organs is still unclear. In addition, during the later stages of life WT1 is diVerentially expressed in certain mammalian cell types. In particular, proper kidney function seems to be regulated by WT1 [3]. Given these multiple functions of WT1, it comes as no surprise that this protein is also implicated in many disorders, diseases and abnormal growth. WT1 is known to be responsible for the tumorigenesis of an early childhood renal neoplasm, Wilm’s Tumor, which arises as a consequence of inactivation of both alleles of the WT1 gene. Other defects in this gene relate to disorders such as the Denys–Drash [6], WAGR [2], and Frasier syndromes [7], all of which carry a common character of malformation of the

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genito-urinary system. WT1 mutations have also been implicated in several acute leukemias [8,9], while altered expression of WT1 has recently been detected in other cancer types such as human breast cancer, non-small cell lung cancer [10–12] and more. Even though WT1 was cloned more then 10 years ago there is still controversy as to what is its essential biochemical function? Initial experiments all pointed towards WT1 being a classical tumor suppressor gene whose function was to regulate the transcription of genes involved particularly in proliferation. [13–15] However, a role in transactivation seems to be of much more likely physiological relevance. One example of this activity is the transactivation of the BclII gene, which plays a pivotal role in inhibiting apoptosis [16]. Another example is the direct activation of the oncogene p21 gene, leading to G1 cell cycle arrest [17]. This is consistent with WT1’s role as a tumor suppressor. In addition, it has also been postulated that there might be an additional function for WT1 as a post-transcriptional regulator. From studies on the cellular location of WT1 it was shown that it is a nuclear protein which has not only a diVuse staining pattern (which is characteristic for transcription factors) but that it is also present in speckles, which is more characteristic of splicing factors [18–20]. WT1 contains two diVerent domains. The C-terminal domain contains 4 zinc Wngers of the “Krüppel” type. It is a member of the EGR (early growth response) gene 1 class of zinc Wngers. The N-terminal domain contains a Q/P-rich trans-regulatory domain, a dimerization domain and a putative RNA binding domain. The WT1 gene comprises 10 exons, encoding a complex pattern of mRNA species. Four distinct transcripts are expressed, reXecting the presence or absence of two alternative splices. [21] One splice variant includes or excludes a three amino acid insert (WT1 + KTS or WT1 ¡ KTS) between Zn Wngers 3 and 4, whereas alternative use of exon 5 results in the presence or absence of 17 amino acids in the amino-terminal domain of WT1. Further complexity arises from RNA editing and use of alternative translation start sites. In total, mammalian genes can encode up to 24 protein isoforms of WT1 [22]. WT1 isoforms bind to DNA and RNA sequences in a sequence-speciWc manner. The addition of 3 amino acids (KTS) by exon 9 between zinc Wngers 3 and 4 changes the binding aYnity. WT1 (¡KTS) has high binding aYnity to putative GC-rich DNA sequences, particularly to the ones that resemble the EGR1 consensus-binding site, 5⬘-GCG-T /GGG-GCG-3⬘. [23,24] In turn, WT1 (+KTS) has noticeably lower binding aYnity to these sequences and prefers more dissimilar sequences, which are not well deWned. Unlike WT1 (¡KTS), WT1 (+KTS) has been demonstrated to have a great aYnity to RNA [23,25]. The vast Weld of impact and multi-functionality of WT1 has spurred considerable interest in the physico- and biochemical characteristics of this protein. A large amount of published evidence exists on the WT1 pathology, the biology of the WT1 gene and the preliminary DNA binding properties of its protein products [26,27]. A number of

NMR studies have been performed on the C-terminal domain of WT1 only [28]. These studies gave indications as to why the KTS fragment abrogated DNA binding. However, no structural information at atomic level exists to this day and no structural investigations have been carried out on either the whole protein or only the N-terminal domain. This paper pioneers this research, presenting data on overexpression and puriWcation of two recombinant proteins, 6HIS-ZN¡wt1 and 6HIS-ZN+wt1, which correspond to the C-terminal region of human WT1 (¡KTS) and WT1 (+KTS), respectively, for the purpose of crystallization and subsequent structure determination. We also describe a series of tests performed to identify the proteins and study their stability and DNA binding characteristics. These tests can be useful for post-expression studies of other DNA binding proteins in general and zinc Wnger proteins in particular. Materials and methods Materials Expression hosts Escherichia coli BL21-CodonPlus (DE3)-RIL and E. coli RosettaBlue (DE3) were purchased from Stratagene (USA) and Novagen (USA), respectively. Cloning host E. coli TOP-10 and cloning system Zero Blunt TOPO PCR Cloning were purchased from Invitrogen (USA). Expression vectors pETM-11 and pETM-30 were provided by the EMBL protein expression and puriWcation core facility in Heidelberg, Germany (http://www-db. embl.de). Another expression vector, pCDF-1b, was purchased from Novagen. Restriction enzymes used throughout the whole investigation were from New England Bio Labs (Beverly, USA). PCR reagents together with DNA ladders were from Fermentas. WT1 antibodies used for Western blot were supplied by Santa Cruz Biotech (USA). Materials used for protein puriWcation were purchased from Amersham Biosciences (Uppsala, Sweden). All other chemicals and reagents including LB growth media are of the highest quality and purity. Methods Cloning and construction of plasmids PCR was carried out for ampliWcation of the C-terminus of WT1 (amino acids 312–449) containing the four zinc Wngers. Full-length cDNA fragments of WT1 (¡KTS) and WT1 (+KTS) were used as templates. A set of primers (forward: 5⬘-CCATGGGGTCGGCATCTGAGA-3⬘, reverse: 5⬘-TCAAAGCGCCAGCTGGAGTT-3⬘), and Pfu Turbo DNA polymerase (Promega) were used in the reaction. The forward primer introduced the initial Met and a NcoI restriction site, whereas the reverse primer introduced a HindIII restriction site downstream of the stop codon. AmpliWed DNA fragments ZN¡wt1 and ZN+wt1 were puriWed from 0.8% agarose gel. Without digesting with

E. Nurmemmedov, M. Thunnissen / Protein Expression and PuriWcation 46 (2006) 379–389

A1

Kan

381

lacI

f1 origin

T7 p

MCS

A2

Kan

NcoI

TEV-site

GST HIS-tag lacI

f1 origin

T7 p MCS

A3

Sm

NcoI

f1 origin

TEV-site

linker

HIS-tag

lacI

T7 p

NcoI

Ek-site MCS

S-Tag

HIS-tag

B

MKHHHHHHPMSDYDIPTTENLYFEGAMGSASETSEKRPFMCATPGCNKRYFKLS HLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKF SRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQ LAL

C

Theoretical pI/M wf or 6HIS-ZN+wt1:9 .93/ 19,7 kDa Theoretical pI/M wf or 6HIS-ZN-wt1:9 .89/ 19,4 kDa

Fig. 1. (A1, A2, and A3) Characteristics of the expression vectors pETM-30 (6346 bp), pETM-11 (6029 bp), and PCDF-1b (3621 bp), respectively. Pointed gene ends represent the transcription orientation. Abbreviations: Kan, kanamycin resistance gene; f1 origin, origin of replication; MCS, multiple cloning site with restriction sites for XhoI, NotI, EagI (only in pETM-30 and pETM-11), HindIII, SalI, BamHI, EcoRI (only in pETM-30 and pETM-11), SacI, KpnI; lacI, gene coding for the lac repressor; T7p, T7 promoter; TEV-site, site encoding the cleavage signal for TEV protease; linker, site encoding the linking region between TEV-site and HIS-tag; HIS-tag, site encoding the hexa-histidine region; Sm, streptomycin/spectinomycin resistance gene; Ek-site, enterokinase restriction site; S-tag, sequence encoding for optional S-tag. All expression constructs (Table 1) are made by insertion into the region between HindIII and NcoI restriction sites. (B) Amino acid sequence of the recombinant proteins. Abbreviations: “M” is the start of the C-terminal segment of wild-type WT1. “KTS” is the region (exon 9) not present in ZN¡wt1. (C) Biochemical properties of the expressed recombinant proteins. Abbreviations: pI, isoelectric point; Mw, molecular weight.

restriction enzymes, the fragments were ligated into a pCRBlunt II-TOPO cloning vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen) according to the manufacturer’s instructions. The ligation products were transformed into a chemically competent E. coli TOP-10 cloning host. Upon extraction of the cloning constructs, they were sequenced to conWrm intactness of the vectors and absence of unwanted mutations using the BigDye Terminator version 3.0 DNA sequencing kit (Applied Biosystems, USA). Cloning constructs were sequentially double-digested with restriction enzymes NcoI and HindIII to obtain the intact ZN¡wt1 and ZN+wt1 fragments with corresponding sticky ends, and ligated into expression vectors pETM-11, pETM-30, and pCDF-1b which were digested with the same enzymes. The expression constructs were transformed into chemically competent E. coli TOP-10 cloning hosts for

ampliWcation. After extraction from the cloning hosts they were veriWed by sequencing as above. They were later transformed into chemically competent expression hosts E. coli BL21-CodonPlus (DE3)-RIL and E. coli RosettaBlue (DE3). Fig. 1 depicts the maps of the expression constructs and shows the amino acid sequences and biochemical properties of the expressed recombinant proteins. Expression of recombinant proteins Recombinant proteins 6HIS-ZN¡wt1, 6HIS-ZN+wt1, 6HIS-GST-ZN¡wt1, 6HIS-GST-ZN+wt1, ZN¡wt1, and ZN+wt1 were overexpressed in glucose-enriched 2YT liquid medium (16 g tryptone, 10 g yeast extract, 5 g NaCl, and 10 g glucose per liter) in the presence of the corresponding antibiotic (Table 1). At an OD600 0.5–0.7 at 37 °C and

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Table 1 Expression vectors, expression constructs, and their tag properties used in the study Vector

Tag

Antibiotic resistance

Construct

Expression

pETM-30 pETM-30 pETM-11 pETM-11 PCDF-1b PCDF-1b

6HIS and GST 6HIS and GST 6HIS 6HIS — —

Kanamycin Kanamycin Kanamycin Kanamycin Streptomycin/spectinomycin Streptomycin/spectinomycin

6HIS-GST-ZN+wt1 6HIS-GST-ZN¡wt1 6HIS-ZN+wt1 6HIS-ZN¡wt1 PCDF-1b-ZN+wt1 PCDF-1b-ZN¡wt1

6HIS-ZN+wt1 6HIS-ZN¡wt1

The rightmost column represents the constructs ultimately chosen for expression studies.

190 rpm (rotations per minute) in shake-Xasks, the cells were induced with 2 mM IPTG and continued to grow for 5 h. The Wnal yield was 3 g of dry cells per 1 L of medium. The cell pellet was washed several times in distilled water and then resuspended in pre-cooled BuVer A: 50 mM Tris– HCl, pH 7.5, 250 mM NaCl, and 10 mM imidazole. It was successively sonicated in ice in several dilute portions for 500 s with 4 s on and 3 s oV times. After centrifugation (17,000 rpm) the supernatant was discarded and the inclusion bodies containing the recombinant proteins were washed three times in BuVer B (BuVer A plus 0.5% Tween 20) to get rid of membrane proteins. The cleaned inclusion bodies were solubilized in BuVer C (BuVer A plus 8.0 M urea) and prepared for Ni–NTA aYnity chromatography. PuriWcation under denaturing conditions Ni–NTA–agarose (Novagen), washed with milliQ water and equilibrated in BuVer C, was added to the solution containing the recombinant proteins in proportion 1 ml resin to 4 mg protein. After gentle shaking for 30 min at room temperature, the mixture was applied to a column in batch mode. The resin was then washed with 10 resin volumes of BuVer C and the protein was eluted with at least 4 volumes of BuVer D (BuVer C containing 400 mM imidazole). Flow-through, wash, and elution fractions were checked for the presence of protein. To prepare for size-exclusion chromatography, the eluted protein was placed into concentrator tubes (MWCO 10 kDa) (Millipore, USA) and brought to a concentration of 50 mg/ml through centrifugation at 4000 rpm. Chromatography under denaturing conditions was performed on a ÄKTA Explorer (Amersham, Sweden) using a Superdex 75 16/60 column. Two hundred and Wfty microliters of the concentrated protein sample was applied to the column equilibrated with BuVer D (BuVer A with 5.0 M urea) for every round of the chromatography process at a Xow rate of 0.5 ml/min. The low injection volume and Xow rate were used to assure maximum resolution of the partially refolded proteins at 5.0 M urea. Protein fractions were collected in 96-well plates. The whole puriWcation process was performed at room temperature. The purest fractions were pooled and treated with 50 mM EDTA (Wnal concentration) for 1 h at room temperature to chelate all of the remaining Ni2+ from the sample. Fig. 2 shows the SDS– PAGE results of the Ni–NTA aYnity and size-exclusion chromatography processes.

Fig. 2. SDS–PAGE analysis of the puriWcation process of 6HIS-ZN+wt1 and 6HIS-ZN¡wt1. Lane M, molecular weight markers; lanes 1–3, Xowthrough, wash and elution of Ni–NTA aYnity chromatography, respectively; lane 4, fraction from size-exclusion chromatography. The arrow shows the protein of interest. Since 6HIS-ZN+wt1 and 6HIS-ZN¡wt1 are very close in molecular weight (19.7 and 19.4 kDa, respectively), only results for 6HIS-ZN¡wt1 are shown.

Stability test and protein quantiWcation PuriWed protein was exposed to a series of systematic stability tests. In the Wrst step, several sets of buVers were prepared with various buVers types and pH values against diVerent concentrations of salts. A stable amount of protein was dialyzed against a constant volume of each of the buVers. After choosing the most promising buVer type, pH, salt, and concentration, a second step was introduced in which these buVer conditions were screened against several diVerent additives, which are known to increase solubility of proteins. Finally, stability (solubility) was assessed both from the amount of refolded protein and the Wnal concentration of the protein. Table 2 shows both the buVers used for these tests and the system for buVer selection. Protein concentration was determined in each step using Bradford protein assay kit (Bio-Rad, USA).

E. Nurmemmedov, M. Thunnissen / Protein Expression and PuriWcation 46 (2006) 379–389

described previously. No serious decrease in refolding eYciency was observed.

Table 2 Scheme of the stability test 1 BuVer, pH

Salt, concentration

Phosphate, pH 6.0 MES, pH 6.5 Tris–HCl, pH 7.0 Tris–HCl, pH 7.5 Tris–HCl, pH 8.0 Bis–Tris propane, pH 8.0 Tris–HCl, pH 8.5 CHES, pH 9.25 CHES, pH 10.0

0 mM NaCl 250 mM NaCl 500 mM NaCl 750 mM NaCl 1 M NaCl 2 M NaCl 3 M NaCl 100 mM Na-malonate 250 mM Na-malonate 500 mM Na-malonate 500 mM NaF

2 BuVer, salt Tris–HCl, pH 7.5, 500 mM NaF

383

SDS–polyacrylamide gel electrophoresis

Other additives, concentration 1-S-octyl--D-thioglucoside, 5 mM n-octyl--D-glucoside, 5 mM Dodecylpoly (ethyleneglycoether) (Thesit) 0.05 mM Cholic acid, 5 mM PEG 400, 10% PEG 1000, 10% Glycerol, 10% v/v

Other constant buVer components: 250 M Zn2SO4, 5 mM DTT, 1 mM GSH, 0.1 mM GSSH, and 0.005% Tween 20 Step 1 (denoted with “1”): each of the buVers on the left is scanned against each of the salts on the right. Step 2 (denoted with “2”): the most favorable condition from step 1 (Tris–HCl, pH 7.5, 500 mM NaF) is scanned against each of the additives on the right. In all of the buVers, conditions shown in the bottom row are kept constant.

Protein refolding The protein in the pooled size-exclusion chromatography fractions was reduced with dithiothreitol (DTT) of Wnal concentration of 100 mM with gentle shaking at room temperature for 30 min. Subsequently, the protein was diluted with BuVer D, brought to a concentration of 0.4–0.5 mg/ml as described previously and placed into dialysis tubes (MWCO 10 kDa). Dialysis was carried out for at least 12 h at 4 °C against 300 volumes of Dialysis BuVer: 50 mM Tris, pH 7.5, 500 mM NaF, 500 M ZnSO4, 5 mM DTT, 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione (GSSH), 0.005% Tween 20, 10% glycerol, and protease inhibitor tablets (Roche, Switzerland). DTT was also added to the buVer such that the Wnal concentration in the system would be 5.0 mM. Upon completion of the process, the dialysate was carefully removed from the dialysis tubes and placed into centrifuge tubes. Protein that failed to refold precipitated and was separated from the refolded protein in the soluble phase via centrifugation at 17,000 rpm. The soluble phase containing the refolded protein was Wltered with 0.2 m Wlter buttons (Millipore) and exposed to a series of further tests. The collected precipitate was kept frozen at ¡20 °C for dialysis recycling. The pellet was regenerated with 8.0 M urea, reduced with 100 mM DTT for 1 h at room temperature and used for dialysis as

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed after each of the above steps for veriWcation of the results, using pre-cast 4–20% SDS–PAGE gels (Bio-Rad) and Mark12 protein weight marker (Invitrogen). Mass spectrometry Clear bands (of pure protein) were carefully cut out of the SDS–PAGE gels and prepared for mass spectroscopy analysis. The Coomassie-stained bands were de-stained 3 times in 50 l of 50% ACN/25 mM NH4HCO3 for 30 min with agitation. The bands were subsequently dried in a speed vacuum for 10–15 min. Dried gel pieces were re-swollen with 30 l of 12.5 ng/l trypsin (Promega, USA) in 50 mM NH4HCO3 solution on ice for 45 min. Trypsin digestion was carried out at 37 °C for at least 12 h. Peptides were extracted from the gel pieces with addition of 30 l 75% ACN, 5% TFA and incubation for 30 min. MALDITOF analysis was performed with the extracted peptide samples at the SWEGENE Facility, Lund, Sweden. Western blot Protein samples were run on a SDS–PAGE gel as described previously. Without Coomassie staining, the gel was processed for Western blot using the ECL Western blot kit. (Amersham). After transferring to nitrocellulose membrane, proteins were probed with primary antibodies, rabbit polyclonal IgG, speciWc to the C-terminal of WT1 (Santa Cruz Biotech, USA) diluted 500–1000 times, with incubation overnight in cold room. The membrane was successively probed with secondary antibody, goat-anti-rabbit IgG (Bio-Rad), conjugated to horseradish peroxidase (HRP) diluted 3000 times for 2 h at room temperature. The membrane was developed with HRP substrate according to manufacturer’s instructions. Circular dichroism spectroscopy Refolded 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 were Wltered and brought to a concentration of 0.5–1.0 mg/ml with dialysis buVer. CD spectra were obtained on a Jasco J-720 spectropolarimeter at 20 °C with the following settings: response 16 s, speed 50 nm/min. Two hundred microliters of 0.5 mg/ml protein solution was placed into 1 mm path length quartz tube and used for measurements with the buVer as control. For each of the samples average of four successive measurements made between wavelengths of 180–260 nm was taken. The secondary structure content was analyzed in comparison with a three-component model [31].

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Electrophoretic mobility shift assay Electrophoretic mobility shift assay was performed to test binding of 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 to doublestranded DNA oligonucleotides containing the Egr1 consensus sequence which has been reported previously [23,24]. Chemically synthesized single-stranded DNA oligonucleotides (5⬘-GTG GCG TGG GCG GCG T GG GCG T-3⬘, 5⬘-CAC GCC CAC GCC GCC CAC GCC A-3⬘) concentrated to 0.5 nmol/l each were heated together in equimolar quantities at 95 °C for 10 min and slowly cooled down to room temperature to obtain double-stranded DNA. Double-stranded DNA was gradually titrated from 0 to 2 nmol in seven reaction mixtures with a constant amount (20 g or 1 nmol) of pure 6HIS-ZN¡wt1 and 6HISZN+wt1. The Wnal reaction volume was brought to 40 l with 5£ binding buVer (25 mM Tris–HCl pH 7.5, 250 mM NaF, 5 mM MgCl2, 3 mM DTT, 50% glycerol, and 10 mM ZnSO4). Poly(dI–dC) of a Wnal concentration of 0.5g/l was added to the reactions separately to address unspeciWc binding of proteins to oligonucleotides. Complexes were allowed to form in a course of 25–30 min at room temperature. Samples were loaded in 50 l volumes into 0.8% agarose gel prepared in TBE buVer (89 mM Tris–HCl, pH 8.4, 89 mM boric acid, and 2 mM EDTA) containing 3 mM ZnSO4. Similarly, processed control experiments with only protein or only DNA were also loaded into the gel. The gel was run in 1£ TBE buVer as above at 4 °C, 75 V for 3 h. Successively, it was stained with SYBR Green EMSA nucleic acid stain (Molecular Probes, USA) according to the manufacturer’s instructions. After visualization on a ChemImager Ready (Alpha Innotech, USA) gel documentation system at 302 nm, a second staining was performed with SYPRO Ruby EMSA protein stain (Molecular Probes) according to the manufacturer’s instructions. The second visualization of the gel was performed as above. Digital records were taken. Another series of similarly processed DNA binding reactions with a DNA to protein molar ratio of 1:1 (1 nmol each) were prepared for both the 6HIS-ZN¡wt1 and 6HISZN+wt1 variant. The reactions were run at Xow rate of 1 ml/ min at room temperature on a Superdex 75 16/60 column equilibrated with a buVer containing 50 mM Tris, pH 7.5,

500 mM NaF, 500 M ZnSO4, 5 mM DTT, 10% glycerol and protease inhibitor tablets. The absorption ratio of aggregated protein over protein–DNA complex was used for estimation of percentage of total active protein (see Table 3), by comparing it to a protein size standard. Results and discussions Cloning, expression, and puriWcation Gradient PCR carried out between 53–67 °C for WT1 zinc Wngers using high-Wdelity DNA polymerase gave single-band ampliWcation for 63–67 °C. The advantage of the pCR-Blunt II-TOPO cloning was that it did not require the inserts to have sticky ends; therefore, ligation was highly eYcient. Expression vectors pETM-11 and pETM-30 are designed to have N-terminal 6HIS-tag and 6HIS-GST-tag, respectively; both of them have a TeV protease cleavage site downstream of these tags. A high level of ligation was also achieved with them. Six single colonies were analyzed for the presence of the insert after every ligation. For the expression of ZN¡wt1 and ZN+wt1, it was decided that puriWcation from inclusion bodies would be the most optimal way. Therefore, a temperature of 37 °C and an IPTG concentration of 2 mM were established via time-course and IPTG titration studies to favor maximum insoluble expression. Choice of the glucose-enriched 2YT medium in comparison to standard liquid LB medium was experimentally proven to enhance both cell growth and the level of expression (data not shown). Expression hosts E. coli RosettaBlue (DE3) and E. coli BL21-CodonPlus (DE3)-RIL, which were used for precautionary reasons, gave no signiWcant diVerence in terms of protein yield (data not shown). Therefore, the latter was chosen for expression scale-up experiments since it grows faster in the chosen media. Of the several expression vectors initially used in the study (Table 1), pETM-11 and pETM-30 constructs oVered similar expression levels. However, the pCDF-1b constructs oVered a notably lower expression level, due to low plasmid copy number. Among all of the constructs, pETM-11 oVered the highest refolding eYciency (Table 3). It seems that the 6HIS-tag contributes to solubility and refolding ability of these proteins. However, the bulkiness and

Table 3 PuriWcation of 6HIS-ZN+wt1 and 6HIS-ZN¡wt1 expressed in E. coli Step

a

c

Total protein Ni2+–NTA aYnity chromatography Size-exclusion chromatography Refolding Protein activity after refoldingd a b c d

6HIS-ZN¡wt1

6HIS-ZN+wt1 b

Weight (mg)

Yield (%)

Purity (%)

Weighta (mg)

Yieldb (%)

Purity (%)

20 8 6.3 4.1–5.0 795%

100 40 31.5 20.5–25

32 76 96 96

18 8.5 6.4 4.1–5.1 795%

100 47 36 23–28.3

35 73 97 97

Amount obtained after completion of the individual puriWcation process. Yields are derived based on the total protein amount. Total protein is that obtained from 1 L of expression culture. Percentage obtained from the total amount of refolded protein.

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complexity of the GST-tag retards the refolding eYciency of the protein it is tagged to. Thus, the pETM-11 constructs (only 6HIS-tag) were chosen for expression scale-up experiments. TeV cleavage of the 6HIS-tag was performed under several conditions; however, it did not improve the eYciency enough for scale-up. Therefore, the 6HIS-tag was left uncleaved for the initial crystallization trials. The sequence of post-expression experiments was chosen with maximum protein purity and yield in mind: aYnity puriWcation, size-exclusion chromatography and then refolding (Table 3). Size-exclusion chromatography was chosen as the second step of puriWcation because Ni–NTA aYnity puriWcation did not yield suYciently pure protein. Since the proteins displayed very instable behavior (aggregation and precipitation) in response to concentration prior to gel Wltration, denaturing conditions were preferred throughout the puriWcation process. Thus, protein loss caused by concentration was reduced to a minimum. Stability, buVers, and refolding The choice of buVer suitable for maximum stability/solubility of the protein was the most time-consuming and one of the crucial parts of this investigation. Proper selection of the ionic strength and reducing environment plays a critical role in acquiring stability and activity of DNA binding proteins, especially for the Cys2-His2-type zinc Wnger proteins. [32,33]. Although 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 are not believed to have disulphide bridges, the Zn2+-coordinating cysteines need to be properly reduced to avoid irregular conformations. Expressed 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 initially aggregated and precipitated both at low ionic strengths and low reducing environments; they could not be concentrated beyond 1 mg/ml. However, for crystallization studies considerably higher concentrations are required. Judging from the theoretical pI of t9.95 for both, in combination with the results of the stability tests, the proteins behaved the most stable at buVer pH 7.0–8.5 with an optimum at pH 7.5. Therefore Tris–HCl, pH 7.5, was chosen as buVer for further studies. Ionic strength also played a critical role: stability enhanced as the NaCl concentration increased from 0 to 1 M; concentrations higher than 1 M had no notable eVect. However, the most stability was provided by 500 mM NaF, which was chosen as the source of ionic strength. The choice of NaF was also inXuenced by the fact that Cl¡ ions interfere with the CD spectroscopy measurements, as they create background absorbance. In contrast, F¡ ions do not show this behavior. Among other additives tried, only the addition of 10% glycerol enhanced protein stability; none of the detergents had any recordable eVect. Oxidation of the Zn2+-coordinating cysteines posed one of the obstacles to the study. The insuYcient reducing environment consisting of 5–15 mM -mercaptoethanol that was used in the beginning of this study caused the protein to aggregate and precipitate. It was replaced by 5 mM DTT, which proved to be more eYcient. Refolding of 6HIS-ZN¡wt1 and 6HISZN+wt1 in these buVer conditions gave eYciency in the range

385

50–85%, which is considerably higher than average published Wgures. After refolding an increase in stability was evident, as 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 could be concentrated up to 5–10 mg/ml, respectively. Mass spectroscopy Mass spectroscopy was performed to have a more solid proof of whether the expressed and puriWed proteins were in fact 6HIS-ZN¡wt1 and 6HIS-ZN+wt1. The analysis gave a number of monoisotopic peaks whose masses were used for comparison to a theoretical digestion of the WT1 protein by trypsin, using the MS-Digest program (128.40.158.151/ucsfhtml3.4/msdigest.htm). Protein identiWcation was done by running the results sequence-wise in both the PIUMS (idelnx81.hh.se/bioinf/mass_spectro.html#PIUMS) and the Mascot (www.matrixscience.com) databases. Search parameters included carbamidomethylation of cysteine, possible oxidation of methionine, and one missed cleavage per peptide. For both 6HIS-ZN¡wt1 and 6HIS-ZN+wt1, the highest similarity was found with Wilm’s Tumor protein (Accession No. P19544). Western blot targeting the C-terminal of WT1 additionally conWrmed the identity of the proteins. This gave us enough conWdence to continue to other parts of the study. Secondary structure analysis CD spectra were obtained in order to estimate the secondary structure content of 6HIS-ZN¡wt1 and 6HISZN+wt1 after refolding. According to the 3-component model [31], measurements performed for these proteins in the DNA-free state yielded a pattern indicative of the presence of both -helix and -sheet (Fig. 3). These results are in agreement with the model that these Cys2-His2-type zinc Wnger proteins consist of a single -helix and two antiparallel -sheets [34,35]. The proteins seem to have obtained their major secondary structure elements (-helix and sheet), as can be inferred from the ratio of the positive and negative ellipticity signals in the CD spectra. However, these elements appear disordered in the uncomplexed protein. In the time-course analysis at 20 °C (data not shown), serious degradation of the positive ellipticity signal (while the negative signal stayed largely undisturbed) was observed. This supports the observation that the -helix is structurally unstable and that the linkers between the individual zinc Wngers are dynamically disordered in the DNAfree state and are, thus, susceptible to structural degradation [28–30]. Binding of WT1 to DNA is believed to stabilize the conformation of the protein and make it less susceptible to degradation. Since the CD spectra and timecourse analysis records for the two isoforms look alike, we infer that there are no signiWcant diVerences in terms of secondary structure and behavior between the two isoforms. Assays on the DNA binding activity of the proteins were in support to our assumptions.

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A

20 15 10

CD

5 0 210

220

230

240

250

-5 -10 -15 -20 -25 -30

Wavelength (nm)

-35

B

30

20

10

0

CD

210

220

230

240

250

-10

-20

-30

-40

Wavelength (nm) Fig. 3. CD spectra for 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 (A and B, respectively) in the DNA-free state. Spectra were obtained at 180–260 nm with a protein concentration of 1.0 mg/ml.

DNA binding activity EMSA experiments can provide strong evidence about a protein’s ability to speciWcally bind to a target DNA and, thus, its correct refolding. CD spectroscopy measurements were not performed for the protein–DNA complexes. However, based on structural studies of similar Cys2-His2-type zinc Wngers, [28–30], it was assumed that binding to DNA would complete induction of the well-deWned  fold and that this binding would stabilize it. For the experiment, a double-stranded DNA with two Egr1 consensus sites (5⬘-G CG-TGG-GCG-3⬘) was used as a substrate for both 6HISZN¡wt1 and 6HIS-ZN+wt1. Considering the pI (t9.9 for each) for the diVerent constructs, and the given pH 8.4 of the buVer used to run experiment, both proteins were posi-

tively charged in the DNA-free state. In the given cathodeto-anode experimental setup, both of the proteins migrated upwards in the DNA-free state (total positive charge) and downwards in the complex state (total negative charge). The charge reversal (from positive to negative) is an indirect indication of protein activity. As the amount of the titrated DNA increased, less protein migrated upwards until it completely stopped beyond the molar ratio of 1:1. Altogether, this presents suYcient qualitative evidence that 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 can speciWcally bind to DNA and form stable complexes. Though all of the experimental parameters of EMSA were kept constant, 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 displayed diVerent DNA binding aYnities. While the former gave a shift as early as at a DNA concentration of 5 M, the

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C

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5

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7

6

7

D

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2

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Fig. 4. Electrophoretic mobility shift assay of 6HIS-ZN¡wt1 (A and B) and 6HIS-ZN+wt1 (C and D). (A and C) Gels stained with SYBR Green nucleic acid stain. (B and D) Gels stained with SYPRO Ruby protein stain. Both 6HIS-ZN¡wt1 and 6HIS-ZN+wt1 are titrated with gradually increasing amount of double-stranded DNA containing double binding site. In (A and C), upper and lower arrows represent protein–DNA complex and free DNA, respectively. In (B and D), they indicate free protein and protein–DNA complex respectively. Mobility of complex is considerably retarded in comparison with that of free DNA. Binding aYnity of 6HIS-ZN¡wt1 is visually higher than that of 6HIS-ZN+wt1. DNA-free protein migrates upwards because of a total positive charge in comparison with a total negative charge of DNA–protein complex. Concentration of DNA increases from 0 to 30 M with 5 M steps (from left to right), while concentration of protein remains constant at 25 M.

latter only starts to shift at 15 M. This indicates that 6HISZN¡wt1 has notably higher DNA aYnity than 6HISZN+wt1 (Fig. 4), which is in line with previous theories [23,25]. Since the aim of this study was to qualitatively show the diVerences in binding aYnity between 6HIS-ZN+wt1 and 6HIS-ZN¡wt1, calculation of the dissociation constants (Kd) was not performed at this stage. The double Egr1 binding site in the DNA duplexes used for the EMSA experiments give the possibility for the formation of two types of protein–DNA complexes through diVerent routes. Depending on the ratio of DNA to protein in the reaction mixture, either one or both of the binding sites can be occupied by protein on a single DNA duplex, thus, form-

ing either monomeric or dimeric protein complexes. When DNA concentration is lower than that of the protein, formation of monomeric complexes is favored, while at DNA concentrations equal to or higher than that of protein, formation of dimeric complexes is favored (see Figs. 4 and 5). SYBR Green DNA stain, which is used for staining in the EMSA experiments, is a bulky molecule and it binds in the minor groove (personal correspondence with Invitrogen). We suggest that the intensity of the SYBR Green DNA dye varies in accordance with availability of the minor groove for binding of dye molecules. This phenomenon is not observed for the other isoform, probably because the KTS insert inXuences the formation of dimers negatively.

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port throughout the study. We also thank Dr. Derek Logan for proofreading the manuscript. References

1

2

3

SYBR Green dye molecule Protein molecule DNA duplex DNA strand with one bound protein and dye molecules DNA strand with two bound protein and unbound dye molecules. Fig. 5. Two types of protein–DNA complex formation. (1) One binding site per DNA duplex is occupied by a protein molecule. [Protein]:[DNA] is between 1:0 and 1:2.5. SYBR Green signal is weak due to low DNA concentration. (2) One binding site per DNA duplex is occupied by a protein molecule. [Protein]:[DNA] is between 1:1.7 and 1:1.25. SYBR Green signal is strong due to high DNA concentration and availability of unoccupied minor groove for binding of dye molecules. (3) Both binding sites per DNA duplex are occupied by protein molecules. [Protein]:[DNA] is between 1:1 and 1:0.7. SYBR Green signal is weak due to fully occupied minor groove.

Conclusion WT1 is a zinc Wnger transcription factor that is critically involved in the process of tumorigenesis. Being active at various cellular levels and having numerous targets, WT1 has stimulated a considerable interest in terms of how its threedimensional structure relates to its functions. In this paper we report expression, puriWcation and characterization of two recombinant proteins 6HIS-ZN¡wt1 and 6HIS-ZN+wt1, which map to the C-terminal portions of ¡KTS and +KTS isoforms of WT1, respectively. We have managed to obtain quantities of stable pure protein variants such that crystallization experiments can be initiated. The methodology presented here made it possible to reveal the factors critical to expression, puriWcation, solubility, refolding, and functional activity of the proteins. A pioneering work for crystallization studies, it provides a valuable blueprint, which can be easily applied to study other zinc Wnger proteins. Acknowledgments We are very grateful to our colleagues Huseyin Uysal and Stepan Shipovskov for valuable discussions and sup-

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