Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus

Protein Expression and Purification 77 (2011) 112–117

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Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus Shivani Agarwal, Sunita Kumari Yadav, Aparna Dixit ⇑ Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110 067, India

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Article history: Received 21 November 2010 and in revised form 31 December 2010 Available online 7 January 2011 Keywords: Tpr Six-histidine tag Rattus norvegicus Heterologous expression

a b s t r a c t Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proliferation of normal cells under pathophysiological conditions. In order to evaluate its potential as a pharmaceutical target, the protein must be produced and purified in sufficiently high yields. In the present study, we report the high level expression of Tpr protein of R. norvegicus employing heterologous host, Escherichia coli, to permit its structural characterization in great detail. We here demonstrate that the Tpr protein was expressed in soluble form and approximately 90 mg/L of the purified protein at the shake flask level could be achieved to near homogeneity using single step-metal chelate affinity chromatography. The amino acid sequence of the protein was confirmed by mass spectroscopic analysis. The highly unstable and disordered Tpr protein was imparted structural and functional stability by the addition of glycerol and it has been shown that the natively unfolded Tpr protein retains DNA binding ability under these conditions only. Thus, the present study emphasizes the significance of an efficient prokaryotic system, which results in a high level soluble expression of a DNA binding protein of eukaryotic origin. Thus, the present strategy employed for purification of the R. novergicus Tpr protein bypasses the need for the tedious expression strategies associated with the eukaryotic expression systems. Ó 2011 Elsevier Inc. All rights reserved.

Introduction The c-Jun, a product of an early response proto-oncogene and a member of the AP-1 family of transcription factor(s), is involved in a variety of biological processes that influence cell differentiation, proliferation, growth and apoptosis [1–4]. Regulation of c-jun expression under different physiological conditions is achieved by interaction of protein factors with the regulatory sequences present in the upstream region of the c-jun. Earlier studies from our laboratory have resulted in the identification of distinct cis-acting elements and trans-acting factor(s) involved in its positive regulation [5,6]. We have demonstrated the presence of a cis-acting regulatory element spanning 538 to 514 region of c-jun which plays a positive role in c-jun transcription [7]. The protein that interacts with the 538 to 514 region of the c-jun and augments transcription from the c-jun promoter is identified as the Translocated promoter region protein (Tpr1). Since the potential of designing therapeutic drugs targeting transcription factors is well known ⇑ Corresponding author. Fax: +91 11 26742580. E-mail addresses: [email protected], [email protected], adixit7@gmail. com (A. Dixit). 1 Abbreviations used: Tpr, translocated promoter region; EMSA, electrophoretic mobility shift assay; RT, room temperature. 1046-5928/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2011.01.001

[8,9], knowledge about the proteins that interact with the identified cis-acting element and modulate c-jun expression can be exploited for designing small molecule inhibitors to curtail c-jun expression during pathophysiological conditions. The first report on the identification of Tpr (involving 140–230 NH2 terminal residues) in fusion with several proto-oncogenes (ras, raf, met, trc) and the potential of this chimera to induce neoplastic transformation provided evidence to the importance of chromosomal translocations in rendering normal cells tumorigenic [10,11]. Until now, Tpr has been characterized as a nucleocytoplasmic protein and no other function has been assigned to this protein [12]. The nuclear pore complex (NPC) links the inner and outer membranes of the nuclear envelope. These pore complexes at the nuclear periphery facilitate the passive diffusion of ions, metabolites and small molecules across the membrane efficiently [13]. Nuclear pore complex proteins are also known to govern the mRNA export post transcription to the cytoplasm for their subsequent translation. The signal mediated transport of macromolecules across the nuclear membrane via the NPC is a highly regulated process and thus points to an important mechanism for control of gene expression [14,15]. Several nucleoporins contain typical conserved motifs that provide evidence to their probable functions. Unlike Tpr, Nup153, a nucleoporin contains four zinc finger motifs through which it binds to DNA in a zinc-dependent fashion in vitro [16–18]. This is consistent with the proposal that nucleoporins play an

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important role in the chromatin organization and nucleocytoplasmic transport. Similar to the Tpr, Nup107 also possesses leucine zippers at its C-terminus, suggesting that either dimerization or the formation of homo-or heteromers is a requisite for its functioning [19]. However, the actual function of NPC-associated Tpr, occurring ubiquitously in all the cells, remained unknown. Normally by itself Tpr does not possess oncogenic/transforming properties and its role in modulating c-jun transcription remains enigmatic. Therefore, discovery of such genes and their fusion products can be exploited as potential targets for cancer therapy. To gain an insight into the diverse functional and structural aspects of Tpr, the present study was aimed to produce the Tpr protein in sufficient yields and quantity to attain high-resolution structure and for making DNA–protein interaction studies feasible. Materials and methods

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fractions were pooled, dialyzed in 20 mM Tris–HCl, pH 8.0 with and without glycerol (10%), unless otherwise stated and stored at 4 °C for immediate use or 20 °C in 50% glycerol for long term use or until further use. The authenticity of the purified recombinant Tpr protein was confirmed by Western blotting analysis by resolving the purified fraction on 12% SDS–PAG and transferring onto nitrocellulose (NC) membrane. The blot was blocked with 2% BSA in 1  PBS containing 0.05% Tween 20 (PBST) for 2 h at RT followed by three subsequent washes with PBST for 10 min each at RT. The membrane was then incubated with the anti-6-His tag antibody conjugated with alkaline phosphatase. The immunoreactive bands were visualized by the Western blue stabilized substrate solution (Promega, USA). Protein concentration was determined by the method of Lowry et al., [22] using bovine serum albumin as a standard. Antibody generation against the rTpr

Materials Escherichia coli DH5a and M15 strains were obtained from Gibco-BRL, USA and BL21 (kDE3) strain was procured from Novagen, USA. The plasmid harboring the cDNA encoding Tpr in pCMVsport 6.ccdb vector (IMAGE clone ID: 7929511) was obtained from the American Type Culture Collection (ATCC), USA. All the chemicals required for DNA manipulation (restriction enzymes and chemicals) were purchased from New England Biolabs, USA and Promega, USA. The radioactive nucleotide a-32P [dCTP], used in the study was procured from BRIT, India. His-3 polyclonal antibody conjugated with the alkaline phosphatase was procured from Sigma Co., USA. All other chemicals of analytical grade were procured from Sigma Co., USA, unless otherwise stated. DNA manipulations were carried out using standard protocols unless otherwise stated [20]. The oligonucleotides and primers used in the study were synthesized by Sigma Co., USA.

BALB/c mice (4–6 weeks) were used for immunization experiments to raise primary antibodies against recombinant protein. The guidelines prescribed by the Institutional Animal Ethics Committee, JNU, New Delhi, India were followed while handling the animals and the animals used for the project had the approval of the Institutional Animal Ethics Committee (IAEC-JNU Project Code No. 01/2005). Mass spectrometry The specific band corresponding to the Tpr protein was excised from the polyacrylamide gel with a sterile scalpel and digested with trypsin. The digested samples were analyzed by an ultraflex MALDI-TOF–TOF instrument (Bruker, Germany). Peptide mass fingerprinting (PMF) was performed by comparing the masses of identified peptides to NCBI protein database using the MASCOT search engine (http://www.matrixscience.com) as described [21].

Construction of 6His-tagged Tpr clones DNA binding ability of the rTpr using EMSA The full length gene encoding Tpr of Rattus norvegicus was PCR amplified using gene specific primers, [Forward: 50 -CCGGGATCCATGGCGGCGGTGTTGCAGCAAGTGC-30 ; Reverse: 50 -CCGGGTACCTTATTCCATAAGGTCTTTAGATTCTTCC-30 containing BamH I and Kpn I (bold and underlined), respectively, for the ease of cloning the gene] and plasmid pCMVsport 6.ccdb as the template. The amplified tpr fragment digested with BamH I and Kpn I was ligated to pQE-30 and pRSET-A prokaryotic expression vectors digested with the same enzymes. Putative recombinants were screened using restriction enzyme digestion analysis and further integrity of the tpr gene in both the constructs was confirmed by automated dideoxy DNA sequencing (Applied Biosystem Model 393A). The recombinant constructs were designated as pQE-30.tpr and pRSETA.tpr.

Electrophoretic mobility shift assay (EMSA) was carried out using 1 lg of the purified rTpr (dialyzed in buffer with and without glycerol) and 5 ng of the a-32P[dCTP] labeled Jun-25SA (oligonucleotide spanning 538 to 514 region of c-jun, 40,000 cpm) in a final reaction volume of 40 ll at 25 °C for 30 min (unless otherwise mentioned) as described [7]. The complex was loaded onto a pre-electrophoresed 5% non-denaturing PAG and the products were analyzed by autoradiography. Supershift assay was performed by preincubating the rTpr (1 lg) with 1 lg of the anti-rTpr antibody prior to the addition of the labeled Jun-25SA. The complex was resolved on a pre-electrophoresed 5% non-denaturing PAG, and the products were analyzed by autoradiography.

Expression and purification of rTpr in E. coli

Results and discussion

The E. coli BL21 (kDE3) cells harboring the pRSETA.tpr and M15 cells harboring the pQE-30.tpr were induced with 1 mM IPTG and the expression of the recombinant proteins was analyzed 8 h post-induction. The localization analysis for the expressed recombinant protein was performed in the induced cell lysates obtained from both the E. coli hosts, essentially as described earlier [21]. All protein purification steps were performed at 4 °C. The recombinant Tpr protein (rTpr) from both the constructs was purified from the soluble fraction under native conditions [21]. The expressed Tpr protein from both the constructs harboring the hexa-histidine tag at the N-terminus was subsequently purified using Ni2+-NTA affinity chromatography. The purified protein

Cloning, expression and purification of the R. norvegicus recombinant 6His-Tpr The versatile functions associated with Tpr, its localization in various cellular compartments and existence of several isoforms/ homologs has been reported [10–15]. However, the mechanisms involved therein are far from clearly understood. In order to elucidate these functions, pure and functional Tpr is required. Earlier studies demonstrating different functions of Tpr have used purified cDNAs to produce full length Tpr protein using an in vitro coupled transcription and translation system for both human and rat Tpr proteins [23,24]. Earlier attempts to purify the full length human

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A high level expression of the 6-His tagged recombinant Tpr protein was achieved from both the constructs, pQE-30.tpr and pRSETA.tpr a upon induction with IPTG (Figs. 1 and 2, Panel A). A predominant band corresponding to the expected size of the rTpr ( 37 kDa from pQE30-tpr;  41 kDa from pRSETA.tpr) could be observed in total cell lysates of the induced cells (Figs. 1 and 2, lane 2, respectively) indicating an efficient expression of the recombinant Tpr. The expression of rTpr in the uninduced cell lysates of the E. coli BL21(kDE3) cells harboring pRSETA.tpr indicated the leaky expression of the recombinant protein, even in the absence of the inducer, which was not observed in the uninduced cell lysates of E. coli M15 cells harboring pQE-30.tpr. Unlike pRSETA.tpr, the expression of the protein from the pQE-30.tpr construct only upon IPTG induction demonstrated the stringency associated with the M15 expression system. Under identical conditions, expression levels of the recombinant Tpr from both the constructs were also strikingly different with a lower expression observed with the pRSETA-tpr construct in contrast to the pQE-30.tpr construct. Localization analysis for the recombinant proteins from both the constructs revealed that the proteins expressed as soluble proteins (Panel B of Figs. 1 and 2 for pQE-30.tpr and pRSETA.tpr, respectively). The rTpr expressed as soluble protein was purified under native conditions

Tpr from various mammalian tissues, or to produce full length N-or C-terminal domains using bacterial and baculoviral expression systems resulted in lower yields with degradation products [25]. Earlier reports from our laboratory have established the functional significance of the 34 kDa protein, identified as Tpr, in cjun transcription by interaction with an upstream element of the c-jun [7]. To evaluate the role of the Tpr (34 kDa) protein in the positive regulation of c-jun transcription by its interaction with the 538 to 514 region of c-jun, large scale production of the protein through recombinant DNA technology was a prerequisite. Therefore, the present study was undertaken to produce functionally active Tpr with high yields. For this purpose, an open reading frame corresponding to the Tpr protein fragment of R. norvegicus encoding a protein (BC101883) of 339 amino acids (Rn-Tpr of 37 kDa) was cloned as a 6-histidine tagged protein under the control of T5 and T7 promoter in pQE-30 and pRSET-A expression plasmids, respectively. The recombinants were confirmed by restriction analysis and automated DNA sequencing. The cloning of the tpr gene in pRSET-A vector resulted in addition of 33 amino acid residues at the N-terminus including 6His tag, and therefore the recombinant protein would be about 41 kDa.

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97 66 45 31 29 14 Fig. 1. Expression, localization and purification of the 6Histidine tagged recombinant Tpr (rTpr) from pQE30.tpr in E. coli M15 cells. (A) Analysis of expression of rTpr. Lanes 1 and 2 show SDS–PAGE analysis of the uninduced and induced cell cultures of E. coli M15 cells harboring pQE-30.tpr A thick band at the expected size in the induced cell lysates (lane 2, indicated by arrow) indicates high level of expression of the rTpr. Lane ‘M’ shows the migration of protein molecular weight markers (kDa). (B) Localization of expression of the rTpr in E. coli M15 cells. Same proportions of various cellular fractions namely extracellular, periplasmic, cytoplasmic, membraneous and inclusion bodies fractions (lanes 3–7, respectively) of the induced cell lysates were analyzed on 12% SDS–PAGE. Lanes 1 and 2 indicate the cell lysates from uninduced and induced cultures, respectively. A band corresponding to the expected size of the protein can be seen both in periplasmic (lane 4) and cytoplasmic fractions (lane 5). The arrow points to the rTpr. (C) SDS–PAGE analysis of the purified rTpr. The expressed recombinant protein was purified from the soluble fraction using Ni2+-NTA affinity purification. Lanes 1 and 2 show the rTpr purified to near homogeneity after elution with 300 mM imidazole. (D) Immunoblot analysis of the rTpr. The authenticity of the purified recombinant protein was assessed by Western blotting using anti-His antibody. Arrows point to the discrete band at 37 kDa in Lane 1. Lane 2 contained unstained protein molecular weight markers.

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Fig. 2. Expression and purification of the rTpr from pRSETA.tpr in E. coli BL21(kDE3) cells. (A) SDS–PAGE (12%) analysis of the cell lysates from the uninduced (lane 1) and induced cultures (lane 2) of E. coli BL21(kDE3) cells harboring the pRSETA.tpr. A band of expected size of 41 kDa could be seen in the induced cell lysates only. Expression levels were relatively lower than that obtained with E. coli M15 cells harboring pQE30.tpr (compare with Fig. 1A). ‘‘M’ shows the protein molecular weight markers. (B) Localization of the expressed Tpr from pRSETA.tpr construct in E. coli BL21(kDE3). Lanes 1 and 2 represent the cell lysates from uninduced and induced cell lysates, respectively. Lanes 3 and 4 denote the cytoplasmic and inclusion body fractions, respectively. (C) Purification of the rTpr from pRSETA.tpr construct expressed in E. coli BL21(kDE3) expressed recombinant proteins. The recombinant protein was purified from the soluble fraction using Ni2+-NTA affinity purification. Lanes 1 and 2 show the purified rTpr eluted with 300 mM imidazole fraction. (D) Western blot analysis of the 6His-rTpr from pRSETA.tpr construct. The authenticity of the purified protein (lanes 1 and 2) was established by Western blot analysis using anti-His antibody. A single band at the expected size of 41 kDa can be seen. The migration of protein molecular weight standards (M) is shown as kDa. Arrow in all the panels point to the expressed rTpr.

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S. Agarwal et al. / Protein Expression and Purification 77 (2011) 112–117 Table 1 Purification of Rattus norvegicus rTpr. Fraction

Total protein (mg)

Total activity (U)a

Specific activityb

Fold purification

% Yield

Sonication supernatant Purified protein

74 22

17,428 14,460

235 657

1 2.8

100 83

Wet cell pellet (1.40 g) obtained from 250 ml of induced cell culture was sonicated in 12.5 ml of buffer. The sonication supernatant was assayed for Tpr activity and processed for purification. a One unit of rTpr activity is defined as the amount of protein required for complete binding with 0.1 pmol of labeled Jun-25SA under the experimental conditions. b Specific activity is the units per milligram protein.

Fig. 3. MALDI-TOF mass spectrum of the tryptic digest of rTpr. (A) Mass spectrum analysis report of the tryptic digest of the rTpr. (B) The identified protein, score, amino acid sequence coverage and the number of identified peptides are shown. The matched peptides ions in the Tpr sequence (shown in red) are shown in bold and underlined. The amino acid residues highlighted in yellow indicate the putative asparagines glycosylation sites. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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using metal chelate affinity chromatography from both the constructs. As evident from the Figs. 1 and 2, highly pure rTpr could be obtained from both the constructs. (Panel C, lanes 1 and 2). The authenticity of the expressed proteins was established by immunoblotting with anti-His antibody. A single immunoreactive band in the rTpr purified from both the expression systems [E. coli M15 cells harboring pQE-30.tpr and E. coli BL21(kDE3) harboring pRSETA.tpr, Panel D in Figs. 1 and 2, respectively] confirmed the authenticity of the protein. In our studies, it was observed that long term storage of the rTpr resulted in aggregation and degradation of the protein. This is not unexpected as Tpr homologs of human and Sacchromyces cerevisiae have been reported as natively unfolded proteins [26,27]. Often, the use of the recombinant proteins for in vitro studies or as therapeutic agents is usually hampered by protein aggregation during expression, purification, storage, which leads to the concomitant loss of their activity. Also, the proteins frequently aggregate at very high concentrations required for structural studies. However, dialysis of the rTpr against buffer containing 10% glycerol and storage therein prevented this aggregation. This suggests that glycerol provides stability to the protein and is crucial for the protein to maintain its native structure and prevent it from aggregation under physiological conditions. Glycerol is commonly used as a structural stabilizer and it is also considered to be suitable for crystallization of the proteins [28]. This interpretation of the protein stability in glycerol or any other co-solvent might have long term implications in terms of its usage in stabilizing proteins to be used as drug/therapeutic targets. Unlike earlier reports by Hase et al., [26] who reported presence of degradation products of the recombinant human Tpr, no degradation products were seen even in the immunoblot analysis of the rTpr in the present study. Attempts to express different domains of human Tpr in fusion with large partners such as GST have also been made, which require removal of the fusion partner, thus adding an additional step to obtain functional form of Tpr and resulting in lower yields. If the fusion partner is not to be removed, it may affect the biological activity of the protein by masking the active site. Also, use of fusion partner may result in the expression of the recombinant protein as inclusion body and it is difficult to measure refolding yields of biologically active protein [29]. Using present strategy, we could obtain sufficiently high quantities of purified rTpr without any degradation using pQE-30 (Fig. 1C, lanes 1 and 2) and pRSET-A (Fig. 2C, lanes 1 and 2). Approximately, 90 mg/L of purified protein could be obtained using pQE-30 expression system at the shake flask level (Table 1). The pRSET-A expression system gave relatively lower yields (25 mg/L). Since the yield of the rTpr produced from pQE30.tpr were much higher, and also unlike the rTpr produced form pRSETA.tpr, it did not have any extra amino acid residues contributed from the vector, except for 6 histidine residues, this was used for further analysis.

Mass spectrum of 6His-rTpr To determine whether the amino acid sequence of rTpr matches the one predicted from DNA sequencing results, the MALDI-TOF– TOF mass spectra of tryptic digests of recombinant protein was characterized to identify the recombinant protein. Thirty two peptide fragments were identified in mass spectra (Fig. 3). By comparing the masses of identified peptides to the hypothetical tryptic peptides for proteins in non-redundant NCBI database using the MASCOT search engine, rTpr was identified with a score of 97. The identified 32 peptide fragments were matched against the deduced amino acid sequence of the rTpr with 45% sequence coverage.

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F Fig. 4. DNA binding potential of the rTpr using EMSA. (A) EMSA was performed with 5 ng of labeled Jun-25SA and varying amounts of purified rTpr [10 and 20 lg each, dialyzed into buffer without glycerol (lanes 2 and 3) and with glycerol (lanes 4 and 5)]. Lane 1 shows only the radiolabeled oligonucleotide. The figure shows autoradiogram of the complex separated on a 5% non-denaturing PAGE. The ‘‘C’’ and ‘‘F’’ indicate the DNA-rTpr complex and free probe, respectively. (B) Specificity of the rTpr and Jun-25SA complex formation. Lane 1 shows the complex ‘C’ obtained in standard EMSA reaction performed with the rTpr (25 lg) and radiolabeled Jun-25SA (5 ng). Lane 2 indicates the EMSA reaction carried out by addition of the 1 lg of the anti-rTpr-IgG to the preformed rTpr-Jun-25SA complex. ‘S’ represents the supershifted complex.

The 6His-rTpr as a DNA binding protein The Tpr was identified as a positive regulatory protein factor that enhanced c-jun transcription by interacting with the 538 to 514 region of the c-jun promoter. Therefore, to evaluate the DNA binding ability of the rTpr, EMSA using radiolabeled oligonucleotide Jun-25SA encompassing the above region of the c-jun and rTpr was carried out. As shown in Fig. 4, the rTpr in buffer containing glycerol was able to form an intense complex with the radiolabeled Jun-25SA (Fig. 4A, lanes 3 and 4). As expected, the rTpr protein dialyzed in buffer without glycerol failed to bind to DNA in a standard gel shift assay (Fig. 4A, lanes 1 and 2). Therefore, to facilitate the crystallization and to gain an insight into the structural aspects of this novel transcription factor, the rTpr protein, dialyzed and stored in glycerol would be the choice material as the protein exhibited DNA binding activity. It is important to note that the presence of 6-His tag did not affect the binding of rTpr with its recognition sequence. Antibody against the rTpr to assess the specificity of the DNA–protein complex revealed that the interaction of the rTpr to its cognate recognition sequence was indeed specific (Fig. 4B, lane 2). The altered mobility of the protein/DNA complex in a super shift assay reflected the formation of an antibody-protein-DNA ternary complex, (depicted as super-shift ‘S’). It has been reported that post-translational modifications like phosphorylation, acetylation, glycosylation and sumoylation in several transcription factors alters the protein conformation facilitating DNA–protein interaction [30,31]. Unlike other transcription factors, the active recombinant Tpr protein from E. coli retains DNA binding ability, obviating the role of post-translational modifications in the DNA-binding, although three putative N-linked glycosylation sites are present in the protein. Thus, the present study has resulted in soluble and stable expression of the Tpr, a eukaryotic nuclear pore protein, employing prokaryotic expression system, in sufficiently high concentrations which has not been reported before. Conclusions E. coli is a prokaryote and its intrinsic characteristics such as protein processing, protein folding, and post translational

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modifications, differ from those of eukaryotes. Therefore, we made an attempt to purify an eukaryotic protein, Tpr, in E. coli and not in Pichia pastoris, considering the tedious protocol and steps involved in the expression and purification. We were able to demonstrate that the protein was expressed in soluble fraction and was able to elicit the DNA binding ability. For proteins with unknown functions, the detection of the refolding process is often infeasible due to the lack of relevant conformation and activity information of the native proteins. In addition, refolding in vitro often brings about an increase in cost and a reduction in yield, which are fatal deficiencies in industry. Acknowledgments The Council of Scientific and Industrial Research, New Delhi is acknowledged for research fellowship to SA. The authors thank Mr. Amresh Kumar Singh for technical assistance. References [1] P. Angel, M. Karin, The role of Jun, Fos and AP-1 complex in cell proliferation and transformation, Biochim. Biophys. Acta 1072 (1991) 129–157. [2] E. Shaulian, M. Karin, AP-1 in cell proliferation and survival, Oncogene 20 (2001) 2390–2400. [3] E. Shaulian, M. Karin, AP-1 as a regulator of cell life and death, Nat. Cell Biol. 4 (2001) 131–136. [4] W. Jochum, E. Passegue, E.F. Wagner, AP-1 in mouse development and tumorigenesis, Oncogene 20 (2001) 2401–2412. [5] D. Sharma, S. Ohri, A. Dixit, The 148 to 124 region of c-jun interacts with a positive regulatory factor in rat liver and enhances transcription, Eur. J. Biochem. 270 (2003) 181–189. [6] S. Ohri, D. Sharma, A. Dixit, Interaction of an 40 kDa protein from regenerating rat liver with the 148 to 124 region of c-jun complexed with RLjunRP coincides with enhanced c-jun expression in proliferating rat liver, Eur. J. Biochem. 271 (2004) 4892–4902. [7] S. Agarwal, Rajkumar, P. Gupta, A. Dixit, Identification and characterization of a positive regulatory cis-element within the upstream region of c-jun, J. Biochem. 144 (2008) 741–752. [8] D. Kletsas, A.G. Papavassiliou, The therapeutic potential of targeting drugs at transcription factors, Expert Opin. Invest. Drugs 8 (1999) 737–746. [9] D.S. Latchman, Transcription factors as potential targets for therapeutic drugs, Curr. Pharm. Biotechnol. 1 (2000) 57–61. [10] N.R. Soman, P. Correa, B.A. Ruiz, G.N. Wogan, The TPR-MET oncogenic rearrangement is present and expressed in human gastric carcinoma and precursor lesions, Proc. Natl. Acad. Sci. USA 88 (1991) 4892–4896. [11] A. Greco, M.A. Pierotti, I. Bongarzone, S. Pagliardini, C. Lanzi, G. Della Porta, TRK-T1 is a novel oncogene formed by the fusion of TPR and TRK genes in human papillary thyroid carcinomas, Oncogene 7 (1992) 237–242. [12] S. Krull, J. Thyberg, B. Björkroth, H.R. Rackwitz, V.C. Cordes, Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket, Mol. Biol. cell 15 (2004) 4261– 4277.

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