- Email: [email protected]
High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment
Process Biochemistry 45 (2010) 292–296
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment Silvia Pellicer a,b, M. Teresa Bes a,b, Andre´s Gonza´lez a,b, Jose´ L. Neira b,c, M. Luisa Peleato a,b, Marı´a F. Fillat a,b,* a b c
Department of Biochemistry and Molecular and Cell Biology, Spain Biocomputation and Complex Systems Physics Institute (BiFi), University of Zaragoza, Pedro Cerbuna, 12, 50009 Zaragoza, Spain Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez, Alicante, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2009 Received in revised form 2 September 2009 Accepted 29 September 2009
Engineering of DNA-binding domains of regulatory proteins aimed to control gene expression requires a deep knowledge of protein–DNA interactions acquired from structural data on purified species. Most DNA-binding proteins work as dimers establishing multiple protein–protein contacts mainly driven by hydrophobic interactions, being its cleansing a difficult task because of solubility problems. One-step purification of soluble, functional recombinant FurA from the cyanobacterium Anabaena sp. PCC 7120 has been achieved using mild chaotropic conditions. FurA was isolated using a Zn-iminodiacetate chromatography of the crude extract obtained after sonication of Escherichia coli in the presence of 2 M guanidium chloride. CD and 1D NMR spectroscopies demonstrate that FurA conserves the native tertiary structure. Functional analysis reveals FurA ability to recognise and bind target DNAs. We propose that the use of chaotropic agents under mild denaturating conditions might have general application in the purification of DNA-binding proteins and other proteins prone to aggregation. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: High-yield recovery DNA-binding proteins Ferric uptake regulator Guanidinium chloride
1. Introduction The design of DNA-binding proteins aimed to control gene expression constitutes a goal for molecular biologists. In fact, they have important repercussions in gene therapy as regulators of transcriptional levels of genes associated with disease [1,2], in agricultural biotechnology allowing the production of transgenic plants with complex advantageous phenotipes obtained by altering transcription of specific genes [3] or in pharmacology. They are also powerful tools for phenotypic engineering and functional genomic analysis in microorganisms [4] and constitute an essential technology to get new insights into the control of virulence factor and toxin gene expression in pathogenic bacteria [5].
Abbreviations: GdnHCl, guanidinium chloride; PMSF, phenylmethylsulphonyl fluoride; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assays; Px, promoter of the gene X; TCA, tricholoracetic acid; TSP, 3-(trimethylsilyl)propionic acid-d4 sodium SALT; IMAC, Immobilized Metal Ion Affinity Chromatography; IPTG, isopropyl-beta-D-thiogalactopyranoside. * Corresponding author at: Department of Biochemistry and Molecular and Cell Biology, Biocomputation and Complex Systems Physics Institute (BiFi), University of Zaragoza, Pedro Cerbuna, 12, 50009 Zaragoza, Spain. Tel.: +34 976761282; fax: +34 976762123. E-mail address: fi[email protected] (M.F. Fillat). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.09.019
The DNA-binding module in these proteins is, in great measure, responsible for the genes that are to be bound and regulated so that the rational outline of new recognition motifs to target desired DNA sequences requires the knowledge of the recognition code that drives their interactions with DNA. In prokaryotes, the Fur (ferric uptake regulator) superfamily of transcriptional regulators controls a large number of genes involved in general metabolism, electron transport, virulence and the defence against acidic and oxidative stresses [6]. It is generally accepted that Fur binds as a dimer to its target DNA using iron as co-repressor. However, a reducing environment is crucial for optimal activity of several Fur proteins [7,8]. The characterization of Fur paralogues present in the same organism shows that subtle differences in protein sequence lead to distinct functions. Therefore, Fur constitutes a good model to analyse protein–DNA interactions that can contribute to enlarge the range of applications of engineered transcription factors. This task involves the development of a suitable method for producing enough amount of purified, soluble, Fur proteins in order to obtain reliable structural data from the different members of this superfamily of master regulators. The availability of efficient purification methods based on fusion expression systems is certainly one of the reasons why nowadays most advances in artificial transcription factor technology rely chiefly on the DNA-recognition properties of the Cys2-His2 zinc finger DNA-binding domains [9,10]. Also, these domains are
S. Pellicer et al. / Process Biochemistry 45 (2010) 292–296
the most abundant protein motifs in eukaryotes and, although each zinc finger typically recognises only three DNA bases, multiple fingers can be linked in tandem so the resulting multifinger protein can recognise longer sequences. However, studies carried out to evaluate modular strategies for the construction of novel polydactyl zinc finger DNA-binding proteins have shown subtle discrepancies among the models used, which require from both structural and biochemical data to acquire a full understanding [11]. Moreover, transcription factors designed using this technology present some limitations derived from the fact that the structural features of the current zinc fingers could impose restrictions on the type of recognized DNA [12]. Therefore, future developments will take advantage of the incorporation of novel domains of protein–DNA interactions. As a member of the Fur family, cyanobacterial FurA modulates several genes related to processes as diverse as photosynthesis, nitrogen metabolism and cyanotoxin production [13,14]. While regulation of FurA from the nitrogen-fixing cyanobacterium Anabaena PCC 7120 has been extensively investigated, structural studies at atomic detail have been hindered, due to the low purification recovery of recombinant FurA by using previous purification schemes based on heparine and Zn-iminodiacetate chromatographies [15]. Protein solubility is often the bottleneck phase of the protein purification process and low solubility is a major drawback in the detailed structural and functional characterisation of many proteins and isolated protein domains. FurA from Anabaena PCC7120 is prone to oligomerization in solution likely due to the presence of intermolecular disulphide bridges and the involvement of hydrophobic interactions. When FurA is purified using a standard purification protocol an important fraction of the protein remains aggregated [15]. Even though the heterogeneous population of aggregates usually obtained after purification is suitable for EMSA assays, it is not adequate for structural analysis. GdnHCl or arginine has been reported as weakly protein interacting additives that overcome low purification yield and poor protein solubility drawbacks [16,17]. In particular, successful isolation of correctly folded thiol-rich proteins has been accomplished by adding low concentrations of chaotropic agents [18,19]. In this work, we have developed a new method for the purification of FurA based on the use of mild guanidinium chloride treatment that greatly increases protein solubility. We also show that its application to other members of the Fur family allows a rapid, high-recovery single-step purification. Functionality and structure of the purified protein are equivalent to those reported for the purified species using heparin and Zn-iminodiacetate chromatographies [8,15]. We suggest that the purification process described here could be of widespread application to proteins able to bind DNA that are prone to oligomerization.
293
purified protein was dialysed against 10 mM acetic acid/acetate buffer at pH 4. Purified preparations were stored at 20 8C until use. 2.2. Electrophoretic mobility shift assays (EMSA) Binding assays were carried out as described previously [20] in the presence of a 297 bp control unspecific DNA fragment containing the alr0523 gene promoter. 2.3. Ultraviolet, far-UV CD and NMR spectroscopies UV–vis measurements were carried out using a UV–vis double beam Kontron Uvikon 942 spectrophotometer (Japan). Protein concentration was determined using a molar extinction coefficient of 13.760 M1 cm1 at 276 nm. Far-UV circular dichroism (CD) measurements were carried out in a Jasco J-810 spectropolarimeter (Japan) with a scan speed of 50 nm/min with a response time of 2 s; six scans were acquired for each sample. Spectra were corrected by subtracting the proper baseline. The observed ellipticity in millidegrees (u) was converted to mean residue molar ellipticity (u)MR in degree cm2 dmol1 residue1 according to [21]: ðuÞMR ¼
u 10lcN
where l is the pathlength cell (in centimeters), c is the protein concentration (in M), and N is the number of amino acids (151 for FurA). Spectra were acquired at pH = 4.0, at 25 8C. 1 D NMR spectra were recorded on a Bruker Avance DRX-500 spectrometer, working at a 1H frequency of 500.14 MHz, at 25 8C. The NMR samples were concentrated (about 1 mM) by using Amicon devices (Millipore, cut-off molecular weight 10 K). The concentrated protein solution was centrifuged briefly to remove insoluble protein and then transferred to a 5 mm NMR; D2O was added to a final 9:1 H2O:D2O volume. Sample pHs were adjusted at a final value of 4.0. The pH was measured at the beginning and end of each experiment using a Russell glass electrode; no differences were observed between both measurements. Values of the pH reported here represent apparent values of pH, without correction for isotope effects. All the experiments were acquired in 10 mM sodium acetate buffer, pH 4.0. TSP was used as the external chemical shift reference. 1D spectra were acquired using 16K data points, averaged over 1024 scans. The residual water signal was removed by using the WATERGATE sequence [22]. Data were zero-filled to 32K, resolution-enhanced, base line corrected, and processed with the Bruker UXNMR software, working on a PC workstation.
3. Results and discussion 3.1. Single-step protein purification Previous work from our laboratory had shown that FurA exhibits a relatively high conformational stability in the presence of chaotropic agents [21]. Fig. 1 shows that GdnHCl-unfolding of FurA occurs at concentrations higher than 2 M at 25 8C; furthermore the stability of the protein was not modified substantially in the temperature range from 15 to 35 8C [21]. Therefore, we thought of GdnHCl as co-solute to prevent protein aggregation and precipitation during purification. FurA from Anabaena PCC7120 amino acid sequence contains a patch of histidine residues able to interact with metal ions immobilised on an IMAC-affinity column [20]. As the binding of the protein to the
2. Materials and methods 2.1. Protein expression and one-step purification Cloning and overexpression of the furA gene from Anabaena PCC7120 was performed as previously described by Bes et al. [20]. Recombinant FurA was purified from 10 g of Escherichia coli cell paste resuspended in 50 ml of buffer A (0.1 M NaH2PO4, 0.01 M Tris, 2 M guanidine–HCl, pH 8.0) containing 1 mM PMSF. The cell suspension was disrupted by sonication using an ice bath with a Dr Hielscher UP200s sonifier for 10 pulses of 45 s at 190 W. Lysates were clarified twice by centrifugation at 48,000 g for 30 min each time. The resulting supernatant was applied to a 10 ml Zn-iminodiacetate column (Chelating Sepharose Fast Flow; GE Healthcare). ZnSO4 was immobilized as indicated by the manufacturer. The column was first washed with 5 vol. of 0.5 M (NH4)2SO4 in buffer A and then with 35 mM glycine in buffer A. When A280nm was lower than 0.1, FurA was eluted with a 3 vol. linear gradient of 0–1 M imidazole in buffer A. Analysis of the resulting protein was performed by SDS-PAGE using 17% (w/w) polyacrylamide gels. Samples containing GdnHCl were precipitated with TCA prior to electrophoresis. Fractions selected according to their purity and FurA contents were concentrated at room temperature by centrifugation in 10KCentricon devices (Millipore). In order to remove GdnHCl and imidazole, the
Fig. 1. Chemical denaturation curve of FurA at 25 8C. The y-axis shows the average energy (as previously defined [21]) versus the GdnHCl concentration. The midpoint of the curve (according to the procedures described in [21]) is 3.3 0.1 M.
294
S. Pellicer et al. / Process Biochemistry 45 (2010) 292–296
affinity matrix relies on the formation of weak coordinated bonds between metal ions immobilised on the column and histidine residues in the protein, the presence of strong denaturating agents, namely urea or GdnHCl, would not affect their interaction. This advantage could be extended to other soluble recombinant proteins that present aggregation along the purification protocol, and a moderate conformational stability against GdnHCl. If they were artificially His-tagged, increased yield in their purification could be possible in the presence of a chaotrope below the denaturation midpoint. Moreover, in the case of FurA, the heparine column purification step used in the former purification protocol [15] could be removed because the presence of the GdnHCl would help in the solubilization of FurA and in its specific interaction with the IMAC column. Purified recombinant FurA was obtained after a single chromatographic step according to the procedure shown in Fig. 2. Sonication and isolation of crude extract in the presence of buffer A produced a clarified pool of proteins with a high content of soluble FurA. The maximum of the elution peak appeared at 0.25 M imidazol yielding around 4.5 mg of purified protein from 10 g of cell paste. In order to avoid undesirable effects of GdnHCl on SDSPAGE, samples were precipitated with TCA prior to electrophoresis. Fig. 3A shows that FurA is the main protein present in whole cells, and in the crude extract after ultrasonic treatment. Direct application of the clarified crude extract on a Zn-iminodiacetate resin in the presence of GdnHCl at subdenaturating concentration, yields the monomeric form of FurA at high purity. The two main bands observed in lane 4 (Fig. 3A) were unequivocally identified as FurA by Western blot analysis (not shown). The presence of a second band due to different redox status of cysteines is consistently observed in different preparations of cyanobacterial Fur proteins as in the case of other members of the Fur family [7]. An additional advantage of the procedure described in this work is that the presence of GdnHCl allows: (i) protein purification at room
Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purification of recombinant FurA in presence of 2 M GdnHCl. (A) FurA from Anabaena PCC 7120—1: total protein from boiled uninduced cells; 2: total protein from boiled cells induced with 1 mM IPTG for 2 h; 3: crude extract obtained by sonication of cells induced as in lane 2; 4: FurA containing fraction after Zn2+ iminodiacetate chromatography; 5: molecular weight markers. The arrow indicates the presence of the Fur protein. (B) Fur from M. aeruginosa—1: crude extract from IPTG induced cells; 2–4: fractions of purified FurA after Zn2+ iminodiacetate chromatography; 5: molecular weight markers.
temperature, since the stability of the protein was not significantly altered in a wide temperature range, due to its expected low heat capacity [21]; and (ii) to obtain high protein concentration suitable for protein crystallisation screenings (up to at least 5 mg/ml). This process has also been tested during purification of several mutants whose yield was negligible in the absence of chaotrope (data not shown). Furthermore, the purification protocol has been also applied in the isolation of other proteins from the Fur family, namely FurB from Anabaena PCC 7120 and Fur from Microcystis aeruginosa [14,23]. Both species show low amino acid sequence homology with FurA from Anabaena PCC7120 and different function. In the case of Fur from Microcystis aeruginosa the results are as good as for FurA from Anabaena PCC 7120 (Fig. 3B). Both proteins remain completely soluble after removal of GdnHCl through dialysis against 10 mM acetic acid/acetate buffer at pH 4. When dialysis was performed against different buffers, such as 100 M phosphate at pH 7 or 50 mM Tris/HCl at pH 8, visible precipitated sample could be observed. This aggregation can be avoided by adding 0.4 M arginine to the dialysis buffer (data not shown). Since arginine interferes in other studies, such as CD and 1H NMR spectroscopies or, alternatively, in protein interaction studies by using isothermal calorimetry, functionality of Fur was assessed at pH 4. 3.2. Functionality of GdnHCl-FurA
Fig. 2. FurA purification flow chart.
Since the protein preparation had been in contact with a chaotrope along the purification process it was necessary to test its functionality. As shown in Fig. 4, FurA was able to recognise and bind
S. Pellicer et al. / Process Biochemistry 45 (2010) 292–296
295
target DNAs. EMSA assays demonstrated the ability of purified FurA to form a protein–DNA complex with the furA promoter in the presence of a competitor DNA. Identical results were observed using the flavodoxin promoter, PisiB (data not shown). In both cases, assays were performed in the optimal conditions using DTT and Mn2+ to mimic the intracellular reducing environment and the presence of the co-repressor [8]. Moreover, we analysed the interaction between the purified GdnHCl-FurA and the heme group, obtaining spectroscopic and kinetic parameters identical to those reported previously [24] (data not shown). 3.3. Native tertiary and secondary structures of GdnHCl-purified FurA
Fig. 4. Ability of FurA purified in presence of 2 M GdnHCl to specifically recognise and bind furA promoter. EMSA were carried out using 300 nM FurA and 140 nM of each Palr0523 and PfurA DNAs. 1: unspecific Palr0523 and PfurA free DNAs; 2: binding of FurA in the presence of 0.1 mM MnCl2 and 1 mM DTT; 3: as lane 2 without MnCl2; 4: as lane 2, without DTT; 5: in the absence of MnCl2 and DTT.
Finally, in order to know whether GdnHCl affects FurA structure, we compared the far-UV CD and 1D NMR spectra of this protein, with those of FurA purified using our previous protocol based on heparinSepharose and Zn-iminodiacetate chromatographies. Far-UV CD shows minima at 208 and 222 nm (Fig. 5A), which are characteristic of a-helix [25]. The 1D NMR methyl region of the spectra (Fig. 5B) shows the same up-field shifted protons, at 0.3, 0.4 and 0.5 ppm, independently of the presence of 2 M GdnHCl, associated to the presence of tertiary interactions involving methyl residues [26]. Thus, we can be certain that both Fur proteins present the same tertiary functional structure. 4. Conclusion The aim of this study was to find a rapid and efficient method to obtain preparations of the transcriptional regulator Fur suitable for structural studies. These studies will provide information about the recognition code that control interactions of this family of master regulators with DNA. We have taken advantage of the relatively high conformational chemical stability of FurA to use 2 M GdnHCl during the purification procedure to avoid protein aggregation. In any protein purification procedure, at every stage activity is lost. The method described here is very suitable since in a single step we obtain a purified protein that maintains native-like tertiary structure, interactions and functionality at pH 4, and furthermore, the procedure allows larger protein concentration stocks. DNAbinding proteins usually work as dimers establishing protein– protein contacts that make them prone to precipitation during purification processes, therefore we suggest that the use of GdnHCl could be of general application in their purification to obtain large yields of pure soluble proteins. Acknowledgements We thank both reviewers for helpful suggestions and discussion. This work was founded by the Spanish Ministerio de Educacio´n y Ciencia (BFU2006-03454). The work of JLN was supported by Projects SAF2008-05742-C02-01 and CSD-2008-00005 from the Spanish Ministerio de Ciencia e Innovacio´n and by Project ACOMP/ 2009/185 from Generalitat Valenciana. References
Fig. 5. (A) Far-UV CD spectra from FurA from Anabaena obtained according to Herna´ndez et al. [21] (dot-dashed line) and using the procedure described in this work. Both proteins present the minima at 208 and 222 nm characteristics a-helix. (B) High field 1D NMR spectra of FurA purified in presence of 2 M GdnHCl (top) and in the absence of chaotrope, using heparine and Zn2+ iminodiacetate chromatographies (bottom). Measurements were taken at 25 8C using 0.5 mM. FurA in 10 mM D2O-AcH/AcNa buffer at pH 4 and 1 mM DTT. The signal observed in both spectra at 2.0 ppm corresponds to the residual non-deuterated acetic acid.
[1] Rebar EJ, Huang Y, Hickey R, Nath AK, Meoli D, Nath S, et al. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat Med 2002;8:1427–32. [2] Lu Y, Tian C, Danialou G, Gilbert R, Petrof BJ, Karpati G, et al. Targeting artificial transcription factors of utrophin A promoter. Effects on dystrophic pathology and muscle function. J Biol Chem 2008;283:34720–7. [3] Guan X, Stege J, Kim M, Dahmani Z, Fan N, Heifetz P, et al. Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors. Proc Natl Acad Sci USA 2002;99:13296–301. [4] Lee JY, Sung BH, Yu BJ, Lee JH, Lee SH, Kim MS, et al. Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli. Nucl Acids Res 2008;36:1–10.
296
S. Pellicer et al. / Process Biochemistry 45 (2010) 292–296
[5] Gilbert MA, Morton EA, Bundle SF, Samuels DS. Artificial regulation of ospC expression in Borrelia burgdorferi. Mol Microbiol 2007;63:1259–73. [6] Lee JW, Helmann JD. Functional specialization within the Fur family of metallo-regulators. Biometals 2007;20:485–99. [7] Ortiz de Orue´ Lucana D, Schrempf H. The DNA-binding characteristics of the Streptomyces reticuli regulator FurS depend on the redox state of its cysteine residues. Mol Gen Genet 2000;264:341–53. [8] Herna´ndez JA, Lo´pez-Gomollo´n S, Muro-Pastor A, Valladares A, Bes MT, Peleato ML, et al. Interaction of FurA from Anabaena sp. PCC 7120 with DNA: a reducing environment and the presence of Mn2+ are positive effectors in the binding to isiB and furA promoters. Biometals 2006;19:259–68. [9] Dhanasekaran M, Negi S, Sugiura Y. Designer zinc finger proteins: tools for creating artificial DNA-binding functional proteins. Acc Chem Res 2006;39:45– 52. [10] Sera T. Zinc-finger-based artificial transcription factors and their applications. Adv Drug Del Rev 2009;61:513–26. [11] Segal DJ, Beerli RR, Blancafort P, Dreier B, Effertz K, Huber A, et al. Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNAbinding proteins. Biochemistry 2003;42:2137–48. [12] Blancafort P, Segal DJ, Barbas III CF. Designing transcription factor architectures for drug discovery. Mol Pharmacol 2004;66:1361–71. [13] Lo´pez-Gomollo´n S, Herna´ndez JA, Pellicer S, Espinosa V, Peleato ML, Fillat MF. Cross-talk between iron and nitrogen regulatory networks in Anabaena (Nostoc) sp. PCC 7120: identification of overlapping genes in FurA and NtcA regulons. J Mol Biol 2007;374:267–81. [14] Martı´n-Luna B, Sevilla E, Herna´ndez JA, Bes MT, Fillat MF, Peleato ML. Fur from Microcystis aeruginosa binds in vitro promoter regions of the microcystin biosynthesis gene cluster. Phytochemistry 2006;67:876–81. [15] Herna´ndez JA, Bes MT, Fillat MF, Neira JL, Peleato ML. Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC
[16] [17] [18] [19]
[20]
[21]
[22] [23]
[24]
[25] [26]
7119: factors affecting its oligomerization state. Biochem J 2002;366:315– 22. Clark EDB, Schwartz E, Rudolph R. Inhibition of aggregation side reactions during in vitro protein folding. Methods Enzymol 1999;309:217–36. Shiraki M, Kudou S, Fujiwara T, Imanaka M. Biophysical effect of amino acids on the prevention of protein aggregation. J Biochem 2002;132:591–5. Orsini G, Goldberg ME. The renaturation of reduced chymotrypsinogen A in guanidine HCl. Refolding versus aggregation. J Biol Chem 1978;253:3453–8. Morjana NA, McKeone BJ, Gilbert HF. Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase. Proc Natl Acad Sci USA 1993;90:2107–11. Bes MT, Herna´ndez JA, Peleato ML, Fillat MF. Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter. FEMS Microbiol Lett 2001;194:187– 92. ˜ os O, Hurtado-Go´mez E, Bes Herna´ndez JA, Meier J, Barrera FN, Ruiz de los Pan MT, et al. The conformational stability and thermodynamics of Fur A (Ferric Uptake Regulator) from Anabaena sp. PCC 7119. Biophys J 2005;89:4188– 200. Piotto M, Saudek V, Sklenar V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 1993;2:661–5. Herna´ndez JA, Lo´pez-Gomollo´n S, Bes MT, Fillat MF, Peleato ML. Three fur homologues from Anabaena sp. PCC 7120: exploring reciprocal protein-promoter recognition. FEMS Microbiol Lett 2004;236:275–82. Herna´ndez JA, Peleato ML, Fillat MF, Bes MT. Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120. FEBS Lett 2004;577:35–41. Woody RW. Circular dichroism. Methods Enzymol 1995;246:34–71. Wu¨thrich K. NMR of proteins and nucleic acids. New York: Wiley and Sons; 1986.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment Silvia Pellicer a,b, M. Teresa Bes a,b, Andre´s Gonza´lez a,b, Jose´ L. Neira b,c, M. Luisa Peleato a,b, Marı´a F. Fillat a,b,* a b c
Department of Biochemistry and Molecular and Cell Biology, Spain Biocomputation and Complex Systems Physics Institute (BiFi), University of Zaragoza, Pedro Cerbuna, 12, 50009 Zaragoza, Spain Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez, Alicante, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2009 Received in revised form 2 September 2009 Accepted 29 September 2009
Engineering of DNA-binding domains of regulatory proteins aimed to control gene expression requires a deep knowledge of protein–DNA interactions acquired from structural data on purified species. Most DNA-binding proteins work as dimers establishing multiple protein–protein contacts mainly driven by hydrophobic interactions, being its cleansing a difficult task because of solubility problems. One-step purification of soluble, functional recombinant FurA from the cyanobacterium Anabaena sp. PCC 7120 has been achieved using mild chaotropic conditions. FurA was isolated using a Zn-iminodiacetate chromatography of the crude extract obtained after sonication of Escherichia coli in the presence of 2 M guanidium chloride. CD and 1D NMR spectroscopies demonstrate that FurA conserves the native tertiary structure. Functional analysis reveals FurA ability to recognise and bind target DNAs. We propose that the use of chaotropic agents under mild denaturating conditions might have general application in the purification of DNA-binding proteins and other proteins prone to aggregation. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: High-yield recovery DNA-binding proteins Ferric uptake regulator Guanidinium chloride
1. Introduction The design of DNA-binding proteins aimed to control gene expression constitutes a goal for molecular biologists. In fact, they have important repercussions in gene therapy as regulators of transcriptional levels of genes associated with disease [1,2], in agricultural biotechnology allowing the production of transgenic plants with complex advantageous phenotipes obtained by altering transcription of specific genes [3] or in pharmacology. They are also powerful tools for phenotypic engineering and functional genomic analysis in microorganisms [4] and constitute an essential technology to get new insights into the control of virulence factor and toxin gene expression in pathogenic bacteria [5].
Abbreviations: GdnHCl, guanidinium chloride; PMSF, phenylmethylsulphonyl fluoride; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assays; Px, promoter of the gene X; TCA, tricholoracetic acid; TSP, 3-(trimethylsilyl)propionic acid-d4 sodium SALT; IMAC, Immobilized Metal Ion Affinity Chromatography; IPTG, isopropyl-beta-D-thiogalactopyranoside. * Corresponding author at: Department of Biochemistry and Molecular and Cell Biology, Biocomputation and Complex Systems Physics Institute (BiFi), University of Zaragoza, Pedro Cerbuna, 12, 50009 Zaragoza, Spain. Tel.: +34 976761282; fax: +34 976762123. E-mail address: fi[email protected] (M.F. Fillat). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.09.019
The DNA-binding module in these proteins is, in great measure, responsible for the genes that are to be bound and regulated so that the rational outline of new recognition motifs to target desired DNA sequences requires the knowledge of the recognition code that drives their interactions with DNA. In prokaryotes, the Fur (ferric uptake regulator) superfamily of transcriptional regulators controls a large number of genes involved in general metabolism, electron transport, virulence and the defence against acidic and oxidative stresses [6]. It is generally accepted that Fur binds as a dimer to its target DNA using iron as co-repressor. However, a reducing environment is crucial for optimal activity of several Fur proteins [7,8]. The characterization of Fur paralogues present in the same organism shows that subtle differences in protein sequence lead to distinct functions. Therefore, Fur constitutes a good model to analyse protein–DNA interactions that can contribute to enlarge the range of applications of engineered transcription factors. This task involves the development of a suitable method for producing enough amount of purified, soluble, Fur proteins in order to obtain reliable structural data from the different members of this superfamily of master regulators. The availability of efficient purification methods based on fusion expression systems is certainly one of the reasons why nowadays most advances in artificial transcription factor technology rely chiefly on the DNA-recognition properties of the Cys2-His2 zinc finger DNA-binding domains [9,10]. Also, these domains are
S. Pellicer et al. / Process Biochemistry 45 (2010) 292–296
the most abundant protein motifs in eukaryotes and, although each zinc finger typically recognises only three DNA bases, multiple fingers can be linked in tandem so the resulting multifinger protein can recognise longer sequences. However, studies carried out to evaluate modular strategies for the construction of novel polydactyl zinc finger DNA-binding proteins have shown subtle discrepancies among the models used, which require from both structural and biochemical data to acquire a full understanding [11]. Moreover, transcription factors designed using this technology present some limitations derived from the fact that the structural features of the current zinc fingers could impose restrictions on the type of recognized DNA [12]. Therefore, future developments will take advantage of the incorporation of novel domains of protein–DNA interactions. As a member of the Fur family, cyanobacterial FurA modulates several genes related to processes as diverse as photosynthesis, nitrogen metabolism and cyanotoxin production [13,14]. While regulation of FurA from the nitrogen-fixing cyanobacterium Anabaena PCC 7120 has been extensively investigated, structural studies at atomic detail have been hindered, due to the low purification recovery of recombinant FurA by using previous purification schemes based on heparine and Zn-iminodiacetate chromatographies [15]. Protein solubility is often the bottleneck phase of the protein purification process and low solubility is a major drawback in the detailed structural and functional characterisation of many proteins and isolated protein domains. FurA from Anabaena PCC7120 is prone to oligomerization in solution likely due to the presence of intermolecular disulphide bridges and the involvement of hydrophobic interactions. When FurA is purified using a standard purification protocol an important fraction of the protein remains aggregated [15]. Even though the heterogeneous population of aggregates usually obtained after purification is suitable for EMSA assays, it is not adequate for structural analysis. GdnHCl or arginine has been reported as weakly protein interacting additives that overcome low purification yield and poor protein solubility drawbacks [16,17]. In particular, successful isolation of correctly folded thiol-rich proteins has been accomplished by adding low concentrations of chaotropic agents [18,19]. In this work, we have developed a new method for the purification of FurA based on the use of mild guanidinium chloride treatment that greatly increases protein solubility. We also show that its application to other members of the Fur family allows a rapid, high-recovery single-step purification. Functionality and structure of the purified protein are equivalent to those reported for the purified species using heparin and Zn-iminodiacetate chromatographies [8,15]. We suggest that the purification process described here could be of widespread application to proteins able to bind DNA that are prone to oligomerization.
293
purified protein was dialysed against 10 mM acetic acid/acetate buffer at pH 4. Purified preparations were stored at 20 8C until use. 2.2. Electrophoretic mobility shift assays (EMSA) Binding assays were carried out as described previously [20] in the presence of a 297 bp control unspecific DNA fragment containing the alr0523 gene promoter. 2.3. Ultraviolet, far-UV CD and NMR spectroscopies UV–vis measurements were carried out using a UV–vis double beam Kontron Uvikon 942 spectrophotometer (Japan). Protein concentration was determined using a molar extinction coefficient of 13.760 M1 cm1 at 276 nm. Far-UV circular dichroism (CD) measurements were carried out in a Jasco J-810 spectropolarimeter (Japan) with a scan speed of 50 nm/min with a response time of 2 s; six scans were acquired for each sample. Spectra were corrected by subtracting the proper baseline. The observed ellipticity in millidegrees (u) was converted to mean residue molar ellipticity (u)MR in degree cm2 dmol1 residue1 according to [21]: ðuÞMR ¼
u 10lcN
where l is the pathlength cell (in centimeters), c is the protein concentration (in M), and N is the number of amino acids (151 for FurA). Spectra were acquired at pH = 4.0, at 25 8C. 1 D NMR spectra were recorded on a Bruker Avance DRX-500 spectrometer, working at a 1H frequency of 500.14 MHz, at 25 8C. The NMR samples were concentrated (about 1 mM) by using Amicon devices (Millipore, cut-off molecular weight 10 K). The concentrated protein solution was centrifuged briefly to remove insoluble protein and then transferred to a 5 mm NMR; D2O was added to a final 9:1 H2O:D2O volume. Sample pHs were adjusted at a final value of 4.0. The pH was measured at the beginning and end of each experiment using a Russell glass electrode; no differences were observed between both measurements. Values of the pH reported here represent apparent values of pH, without correction for isotope effects. All the experiments were acquired in 10 mM sodium acetate buffer, pH 4.0. TSP was used as the external chemical shift reference. 1D spectra were acquired using 16K data points, averaged over 1024 scans. The residual water signal was removed by using the WATERGATE sequence [22]. Data were zero-filled to 32K, resolution-enhanced, base line corrected, and processed with the Bruker UXNMR software, working on a PC workstation.
3. Results and discussion 3.1. Single-step protein purification Previous work from our laboratory had shown that FurA exhibits a relatively high conformational stability in the presence of chaotropic agents [21]. Fig. 1 shows that GdnHCl-unfolding of FurA occurs at concentrations higher than 2 M at 25 8C; furthermore the stability of the protein was not modified substantially in the temperature range from 15 to 35 8C [21]. Therefore, we thought of GdnHCl as co-solute to prevent protein aggregation and precipitation during purification. FurA from Anabaena PCC7120 amino acid sequence contains a patch of histidine residues able to interact with metal ions immobilised on an IMAC-affinity column [20]. As the binding of the protein to the
2. Materials and methods 2.1. Protein expression and one-step purification Cloning and overexpression of the furA gene from Anabaena PCC7120 was performed as previously described by Bes et al. [20]. Recombinant FurA was purified from 10 g of Escherichia coli cell paste resuspended in 50 ml of buffer A (0.1 M NaH2PO4, 0.01 M Tris, 2 M guanidine–HCl, pH 8.0) containing 1 mM PMSF. The cell suspension was disrupted by sonication using an ice bath with a Dr Hielscher UP200s sonifier for 10 pulses of 45 s at 190 W. Lysates were clarified twice by centrifugation at 48,000 g for 30 min each time. The resulting supernatant was applied to a 10 ml Zn-iminodiacetate column (Chelating Sepharose Fast Flow; GE Healthcare). ZnSO4 was immobilized as indicated by the manufacturer. The column was first washed with 5 vol. of 0.5 M (NH4)2SO4 in buffer A and then with 35 mM glycine in buffer A. When A280nm was lower than 0.1, FurA was eluted with a 3 vol. linear gradient of 0–1 M imidazole in buffer A. Analysis of the resulting protein was performed by SDS-PAGE using 17% (w/w) polyacrylamide gels. Samples containing GdnHCl were precipitated with TCA prior to electrophoresis. Fractions selected according to their purity and FurA contents were concentrated at room temperature by centrifugation in 10KCentricon devices (Millipore). In order to remove GdnHCl and imidazole, the
Fig. 1. Chemical denaturation curve of FurA at 25 8C. The y-axis shows the average energy (as previously defined [21]) versus the GdnHCl concentration. The midpoint of the curve (according to the procedures described in [21]) is 3.3 0.1 M.
294
S. Pellicer et al. / Process Biochemistry 45 (2010) 292–296
affinity matrix relies on the formation of weak coordinated bonds between metal ions immobilised on the column and histidine residues in the protein, the presence of strong denaturating agents, namely urea or GdnHCl, would not affect their interaction. This advantage could be extended to other soluble recombinant proteins that present aggregation along the purification protocol, and a moderate conformational stability against GdnHCl. If they were artificially His-tagged, increased yield in their purification could be possible in the presence of a chaotrope below the denaturation midpoint. Moreover, in the case of FurA, the heparine column purification step used in the former purification protocol [15] could be removed because the presence of the GdnHCl would help in the solubilization of FurA and in its specific interaction with the IMAC column. Purified recombinant FurA was obtained after a single chromatographic step according to the procedure shown in Fig. 2. Sonication and isolation of crude extract in the presence of buffer A produced a clarified pool of proteins with a high content of soluble FurA. The maximum of the elution peak appeared at 0.25 M imidazol yielding around 4.5 mg of purified protein from 10 g of cell paste. In order to avoid undesirable effects of GdnHCl on SDSPAGE, samples were precipitated with TCA prior to electrophoresis. Fig. 3A shows that FurA is the main protein present in whole cells, and in the crude extract after ultrasonic treatment. Direct application of the clarified crude extract on a Zn-iminodiacetate resin in the presence of GdnHCl at subdenaturating concentration, yields the monomeric form of FurA at high purity. The two main bands observed in lane 4 (Fig. 3A) were unequivocally identified as FurA by Western blot analysis (not shown). The presence of a second band due to different redox status of cysteines is consistently observed in different preparations of cyanobacterial Fur proteins as in the case of other members of the Fur family [7]. An additional advantage of the procedure described in this work is that the presence of GdnHCl allows: (i) protein purification at room
Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purification of recombinant FurA in presence of 2 M GdnHCl. (A) FurA from Anabaena PCC 7120—1: total protein from boiled uninduced cells; 2: total protein from boiled cells induced with 1 mM IPTG for 2 h; 3: crude extract obtained by sonication of cells induced as in lane 2; 4: FurA containing fraction after Zn2+ iminodiacetate chromatography; 5: molecular weight markers. The arrow indicates the presence of the Fur protein. (B) Fur from M. aeruginosa—1: crude extract from IPTG induced cells; 2–4: fractions of purified FurA after Zn2+ iminodiacetate chromatography; 5: molecular weight markers.
temperature, since the stability of the protein was not significantly altered in a wide temperature range, due to its expected low heat capacity [21]; and (ii) to obtain high protein concentration suitable for protein crystallisation screenings (up to at least 5 mg/ml). This process has also been tested during purification of several mutants whose yield was negligible in the absence of chaotrope (data not shown). Furthermore, the purification protocol has been also applied in the isolation of other proteins from the Fur family, namely FurB from Anabaena PCC 7120 and Fur from Microcystis aeruginosa [14,23]. Both species show low amino acid sequence homology with FurA from Anabaena PCC7120 and different function. In the case of Fur from Microcystis aeruginosa the results are as good as for FurA from Anabaena PCC 7120 (Fig. 3B). Both proteins remain completely soluble after removal of GdnHCl through dialysis against 10 mM acetic acid/acetate buffer at pH 4. When dialysis was performed against different buffers, such as 100 M phosphate at pH 7 or 50 mM Tris/HCl at pH 8, visible precipitated sample could be observed. This aggregation can be avoided by adding 0.4 M arginine to the dialysis buffer (data not shown). Since arginine interferes in other studies, such as CD and 1H NMR spectroscopies or, alternatively, in protein interaction studies by using isothermal calorimetry, functionality of Fur was assessed at pH 4. 3.2. Functionality of GdnHCl-FurA
Fig. 2. FurA purification flow chart.
Since the protein preparation had been in contact with a chaotrope along the purification process it was necessary to test its functionality. As shown in Fig. 4, FurA was able to recognise and bind
S. Pellicer et al. / Process Biochemistry 45 (2010) 292–296
295
target DNAs. EMSA assays demonstrated the ability of purified FurA to form a protein–DNA complex with the furA promoter in the presence of a competitor DNA. Identical results were observed using the flavodoxin promoter, PisiB (data not shown). In both cases, assays were performed in the optimal conditions using DTT and Mn2+ to mimic the intracellular reducing environment and the presence of the co-repressor [8]. Moreover, we analysed the interaction between the purified GdnHCl-FurA and the heme group, obtaining spectroscopic and kinetic parameters identical to those reported previously [24] (data not shown). 3.3. Native tertiary and secondary structures of GdnHCl-purified FurA
Fig. 4. Ability of FurA purified in presence of 2 M GdnHCl to specifically recognise and bind furA promoter. EMSA were carried out using 300 nM FurA and 140 nM of each Palr0523 and PfurA DNAs. 1: unspecific Palr0523 and PfurA free DNAs; 2: binding of FurA in the presence of 0.1 mM MnCl2 and 1 mM DTT; 3: as lane 2 without MnCl2; 4: as lane 2, without DTT; 5: in the absence of MnCl2 and DTT.
Finally, in order to know whether GdnHCl affects FurA structure, we compared the far-UV CD and 1D NMR spectra of this protein, with those of FurA purified using our previous protocol based on heparinSepharose and Zn-iminodiacetate chromatographies. Far-UV CD shows minima at 208 and 222 nm (Fig. 5A), which are characteristic of a-helix [25]. The 1D NMR methyl region of the spectra (Fig. 5B) shows the same up-field shifted protons, at 0.3, 0.4 and 0.5 ppm, independently of the presence of 2 M GdnHCl, associated to the presence of tertiary interactions involving methyl residues [26]. Thus, we can be certain that both Fur proteins present the same tertiary functional structure. 4. Conclusion The aim of this study was to find a rapid and efficient method to obtain preparations of the transcriptional regulator Fur suitable for structural studies. These studies will provide information about the recognition code that control interactions of this family of master regulators with DNA. We have taken advantage of the relatively high conformational chemical stability of FurA to use 2 M GdnHCl during the purification procedure to avoid protein aggregation. In any protein purification procedure, at every stage activity is lost. The method described here is very suitable since in a single step we obtain a purified protein that maintains native-like tertiary structure, interactions and functionality at pH 4, and furthermore, the procedure allows larger protein concentration stocks. DNAbinding proteins usually work as dimers establishing protein– protein contacts that make them prone to precipitation during purification processes, therefore we suggest that the use of GdnHCl could be of general application in their purification to obtain large yields of pure soluble proteins. Acknowledgements We thank both reviewers for helpful suggestions and discussion. This work was founded by the Spanish Ministerio de Educacio´n y Ciencia (BFU2006-03454). The work of JLN was supported by Projects SAF2008-05742-C02-01 and CSD-2008-00005 from the Spanish Ministerio de Ciencia e Innovacio´n and by Project ACOMP/ 2009/185 from Generalitat Valenciana. References
Fig. 5. (A) Far-UV CD spectra from FurA from Anabaena obtained according to Herna´ndez et al. [21] (dot-dashed line) and using the procedure described in this work. Both proteins present the minima at 208 and 222 nm characteristics a-helix. (B) High field 1D NMR spectra of FurA purified in presence of 2 M GdnHCl (top) and in the absence of chaotrope, using heparine and Zn2+ iminodiacetate chromatographies (bottom). Measurements were taken at 25 8C using 0.5 mM. FurA in 10 mM D2O-AcH/AcNa buffer at pH 4 and 1 mM DTT. The signal observed in both spectra at 2.0 ppm corresponds to the residual non-deuterated acetic acid.
[1] Rebar EJ, Huang Y, Hickey R, Nath AK, Meoli D, Nath S, et al. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat Med 2002;8:1427–32. [2] Lu Y, Tian C, Danialou G, Gilbert R, Petrof BJ, Karpati G, et al. Targeting artificial transcription factors of utrophin A promoter. Effects on dystrophic pathology and muscle function. J Biol Chem 2008;283:34720–7. [3] Guan X, Stege J, Kim M, Dahmani Z, Fan N, Heifetz P, et al. Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors. Proc Natl Acad Sci USA 2002;99:13296–301. [4] Lee JY, Sung BH, Yu BJ, Lee JH, Lee SH, Kim MS, et al. Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli. Nucl Acids Res 2008;36:1–10.
296
S. Pellicer et al. / Process Biochemistry 45 (2010) 292–296
[5] Gilbert MA, Morton EA, Bundle SF, Samuels DS. Artificial regulation of ospC expression in Borrelia burgdorferi. Mol Microbiol 2007;63:1259–73. [6] Lee JW, Helmann JD. Functional specialization within the Fur family of metallo-regulators. Biometals 2007;20:485–99. [7] Ortiz de Orue´ Lucana D, Schrempf H. The DNA-binding characteristics of the Streptomyces reticuli regulator FurS depend on the redox state of its cysteine residues. Mol Gen Genet 2000;264:341–53. [8] Herna´ndez JA, Lo´pez-Gomollo´n S, Muro-Pastor A, Valladares A, Bes MT, Peleato ML, et al. Interaction of FurA from Anabaena sp. PCC 7120 with DNA: a reducing environment and the presence of Mn2+ are positive effectors in the binding to isiB and furA promoters. Biometals 2006;19:259–68. [9] Dhanasekaran M, Negi S, Sugiura Y. Designer zinc finger proteins: tools for creating artificial DNA-binding functional proteins. Acc Chem Res 2006;39:45– 52. [10] Sera T. Zinc-finger-based artificial transcription factors and their applications. Adv Drug Del Rev 2009;61:513–26. [11] Segal DJ, Beerli RR, Blancafort P, Dreier B, Effertz K, Huber A, et al. Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNAbinding proteins. Biochemistry 2003;42:2137–48. [12] Blancafort P, Segal DJ, Barbas III CF. Designing transcription factor architectures for drug discovery. Mol Pharmacol 2004;66:1361–71. [13] Lo´pez-Gomollo´n S, Herna´ndez JA, Pellicer S, Espinosa V, Peleato ML, Fillat MF. Cross-talk between iron and nitrogen regulatory networks in Anabaena (Nostoc) sp. PCC 7120: identification of overlapping genes in FurA and NtcA regulons. J Mol Biol 2007;374:267–81. [14] Martı´n-Luna B, Sevilla E, Herna´ndez JA, Bes MT, Fillat MF, Peleato ML. Fur from Microcystis aeruginosa binds in vitro promoter regions of the microcystin biosynthesis gene cluster. Phytochemistry 2006;67:876–81. [15] Herna´ndez JA, Bes MT, Fillat MF, Neira JL, Peleato ML. Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC
[16] [17] [18] [19]
[20]
[21]
[22] [23]
[24]
[25] [26]
7119: factors affecting its oligomerization state. Biochem J 2002;366:315– 22. Clark EDB, Schwartz E, Rudolph R. Inhibition of aggregation side reactions during in vitro protein folding. Methods Enzymol 1999;309:217–36. Shiraki M, Kudou S, Fujiwara T, Imanaka M. Biophysical effect of amino acids on the prevention of protein aggregation. J Biochem 2002;132:591–5. Orsini G, Goldberg ME. The renaturation of reduced chymotrypsinogen A in guanidine HCl. Refolding versus aggregation. J Biol Chem 1978;253:3453–8. Morjana NA, McKeone BJ, Gilbert HF. Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase. Proc Natl Acad Sci USA 1993;90:2107–11. Bes MT, Herna´ndez JA, Peleato ML, Fillat MF. Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter. FEMS Microbiol Lett 2001;194:187– 92. ˜ os O, Hurtado-Go´mez E, Bes Herna´ndez JA, Meier J, Barrera FN, Ruiz de los Pan MT, et al. The conformational stability and thermodynamics of Fur A (Ferric Uptake Regulator) from Anabaena sp. PCC 7119. Biophys J 2005;89:4188– 200. Piotto M, Saudek V, Sklenar V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 1993;2:661–5. Herna´ndez JA, Lo´pez-Gomollo´n S, Bes MT, Fillat MF, Peleato ML. Three fur homologues from Anabaena sp. PCC 7120: exploring reciprocal protein-promoter recognition. FEMS Microbiol Lett 2004;236:275–82. Herna´ndez JA, Peleato ML, Fillat MF, Bes MT. Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120. FEBS Lett 2004;577:35–41. Woody RW. Circular dichroism. Methods Enzymol 1995;246:34–71. Wu¨thrich K. NMR of proteins and nucleic acids. New York: Wiley and Sons; 1986.