Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor.

The transcription factor PDX1 is a master regulator essential for proper development of the pancreas, duodenum and antrum. Furthermore, it is an indispensable reprogramming factor for the derivation of human ß-cells, and recently, it has been identified as a tumor suppressor protein in gastric cancer. Here, we report the soluble expression and purification of the full-length human PDX1 protein from a heterologous system. To achieve this, the 849 bp coding sequence of the PDX1 gene was first codon-optimized for expression in Escherichia coli (E. coli). This codon-optimized gene sequence was fused to a protein transduction domain, a nuclear localization sequence, and a His-tag, and this insert was cloned into the protein expression vector for expression in E. coli strain BL21(DE3). Next, screening and identification of the suitable gene construct and optimal expression conditions to obtain this recombinant fusion protein in a soluble form was performed. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Importantly, the secondary structure of the protein was retained after purification. Further, this recombinant PDX1 fusion protein was applied to human cells and showed the ability to enter the cells as well as translocate to the nucleus. This recombinant tool can be used as a safe tool and can potentially replace its genetic and viral forms in the reprogramming process to induce a ß-cell-specific transcriptional profile in an integration-free manner. Additionally, it can also be used to elucidate its role in cellular processes and for structural and biochemical studies.

Homeobox proteins are potential biomarkers and therapeutic targets in gastric cancer: a systematic review and meta-analysis.

BACKGROUND: An increasing number of studies have described the aberrant expression of homeobox (HOX) proteins in gastric cancer (GC), which is critically associated with the prognosis and clinicopathological characteristics of GC. This study was conducted to investigate the clinical value and action mechanisms of HOX proteins in GC. METHODS: A comprehensive search of PubMed, Embase, Web of Science and Cochrane Library was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. The pooled hazard ratio (HR) with its 95% confidence interval (95% CI) and the pooled odds ratio (OR) with its 95% CI were used to assess the effect of HOX protein expression on the prognosis and clinicopathological features of GC, respectively. RESULTS: Nineteen studies containing 3775 patients were selected for this study. Heterogeneity among HRs of overall survival (OS) was markedly high (I2 = 90.5%, p = 0.000). According to the subgroup analysis, increased expression of HOX protein in the downregulated subgroup was associated with a good prognosis for patients with GC (pooled HR: 0.46, 95% CI: 0.36-0.59, I2 = 3.1%, p = 0.377), while overexpression of HOX protein in the upregulated subgroup was correlated with a reduced OS (pooled HR: 2.59, 95% CI: 1.79-3.74, I2 = 73.5%, p = 0.000). The aberrant expression of HOX protein was crucially related to the TNM stage, depth of tumour invasion, tumour size, lymph node metastasis, distant metastasis, vascular invasion, histological differentiation and Lauren classification in patients with GC. In addition, the molecular mechanisms by which HOX proteins regulate tumorigenesis and development of GC were also explored. CONCLUSIONS: HOX proteins play vital roles in GC progression, which might serve as prognostic markers and therapeutic targets for GC.

Rapid and efficient purification of Drosophila homeodomain transcription factors for biophysical characterization.

Homeodomain transcription factors (HD TFs) are a large class of evolutionarily conserved DNA binding proteins that contain a basic 60-amino acid region required for binding to specific DNA sites. In Drosophila melanogaster, many of these HD TFs are expressed in the early embryo and control transcription of target genes in development through their interaction with cis-regulatory modules. Previous studies where some of the Drosophila HD TFs were purified required the use of strong denaturants (i.e. 6 M urea) and multiple chromatography columns, making the downstream biochemical examination of the isolated protein difficult. To circumvent these obstacles, we have developed a streamlined expression and purification protocol to produce large yields of Drosophila HD TFs. Using the HD TFs FUSHI-TARAZU (FTZ), ANTENNAPEDIA (ANTP), ABDOMINAL-A (ABD-A), ABDOMINAL-B (ABD-B), and ULTRABITHORAX (UBX) as examples, we demonstrate that our 3-day protocol involving the overexpression of His6-SUMO fusion constructs in E. coli followed by a Ni2+-IMAC, SUMO-tag cleavage with the SUMO protease Ulp1, and a heparin column purification produces pure, soluble protein in biological buffers around pH 7 in the absence of denaturants. Electrophoretic mobility shift assays (EMSA) confirm that the purified HD proteins are functional and nuclear magnetic resonance (NMR) spectra confirm that the purified HDs are well-folded. These purified HD TFs can be used in future biophysical experiments to structurally and biochemically characterize how and why these HD TFs bind to different DNA sequences and further probe how nucleotide differences contribute to TF-DNA specificity in the HD family.

Solution Ensemble of the C-Terminal Domain from the Transcription Factor Pdx1 Resembles an Excluded Volume Polymer.

The pancreatic and duodenal homeobox 1 (Pdx1) is an essential pancreatic transcription factor. The C-terminal intrinsically disordered domain of Pdx1 (Pdx1-C) has a heavily biased amino acid composition; most notably, 18 of 83 residues are proline, including a hexaproline cluster near the middle of the chain. For these reasons, Pdx1-C is an attractive target for structure characterization, given the availability of suitable methods. To determine the solution ensembles of disordered proteins, we have developed a suite of 13C direct-detect NMR experiments that provide high spectral quality, even in the presence of strong proline enrichment. Here, we have extended our suite of NMR experiments to include four new pulse programs designed to record backbone residual dipolar couplings in a 13C,15N-CON detection format. Using our NMR strategy, in combination with small-angle X-ray scattering measurements and Monte Carlo simulations, we have determined that Pdx1-C is extended in solution, with a radius of gyration and internal scaling similar to that of an excluded volume polymer, and a subtle tendency toward a collapsed structure to the N-terminal side of the hexaproline sequence. This structure leaves Pdx1-C exposed for interactions with trans-regulatory co-factors that contribute with Pdx1 to transcription control in the cell.

Separating full-length protein from aggregating proteolytic products using filter flow-through purification.

Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation.

Expression patterns of homeobox genes in the mouse vomeronasal organ at postnatal stages.

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer.

NANOG is multiply phosphorylated and directly modified by ERK2 and CDK1 in vitro.

NANOG is a divergent homeobox protein and a core component of the transcriptional circuitry that sustains pluripotency and self-renewal. Although NANOG has been extensively studied on the transcriptional level, little is known regarding its posttranslational regulation, likely due to its low abundance and challenging physical properties. Here, we identify eleven phosphorylation sites on endogenous human NANOG, nine of which mapped to single amino acids. To screen for the signaling molecules that impart these modifications, we developed the multiplexed assay for kinase specificity (MAKS). MAKS simultaneously tests activity for up to ten kinases while directly identifying the substrate and exact site of phosphorylation. Using MAKS, we discovered site-specific phosphorylation by ERK2 and CDK1/CyclinA2, providing a putative link between key signaling pathways and NANOG.

Challenges of "sticky" co-immunoprecipitation: polyalanine tract protein-protein interactions.

Co-immunoprecipitation (Co-IP) (followed by immunoblotting) is a technique widely used to characterize specific protein-protein interactions. Investigating interactions of proteins containing "sticky" polyalanine (PolyA) tracts encounters difficulties using conventional Co-IP procedures. Here, we present strategies to specifically capture proteins containing these difficult PolyA tracts, enabling subsequent robust detection of interacting proteins by Co-IP.

Isolation of Hox cluster genes from insects reveals an accelerated sequence evolution rate.

Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution.

V(D)J recombination: mechanisms of initiation.

V(D)J recombination assembles immunoglobulin and T cell receptor genes during lymphocyte development through a series of carefully orchestrated DNA breakage and rejoining events. DNA cleavage requires a series of protein-DNA complexes containing the RAG1 and RAG2 proteins and recombination signals that flank the recombining gene segments. In this review, we discuss recent advances in our understanding of the function and domain organization of the RAG proteins, the composition and structure of RAG-DNA complexes, and the pathways that lead to the formation of these complexes. We also consider the functional significance of RAG-mediated histone recognition and ubiquitin ligase activities, and the role played by RAG in ensuring proper repair of DNA breaks made during V(D)J recombination. Finally, we propose a model for the formation of RAG-DNA complexes that involves anchoring of RAG1 at the recombination signal nonamer and RAG2-dependent surveillance of adjoining DNA for suitable spacer and heptamer sequences.

Hsp90 can accommodate the simultaneous binding of the FKBP52 and HOP proteins.

The regulation of steroidogenic hormone receptor-mediated activity plays an important role in the development of hormone-dependent cancers. For example, during prostate carcinogenesis, the regulatory function played by the androgen receptor is often converted from a growth suppressor to an oncogene thus promoting prostate cancer cell survival and eventual metastasis. Within the cytoplasm, steroid hormone receptor activity is regulated by the Hsp90 chaperone in conjunction with a series of co-chaperone proteins. Collectively, Hsp90 and its binding associates form a large heteromeric complex that scaffold the fully mature receptor for binding with the respective hormone. To date our understanding of the interactions between Hsp90 with the various TPR domain-containing co-chaperone proteins is limited due to a lack of available structural information. Here we present the stable formation of Hsp90(2)-FKBP52(1)- HOP(2) and Hsp90(2)-FKBP52(1)-p23(2)-HOP(2) complexes as detected by immunoprecipitation, time course dynamic light scattering and electron microscopy. The simultaneous binding of FKBP52 and HOP to the Hsp90 dimer provide direct evidence of a novel chaperone sub-complex that likely plays a transient role in the regulation of the fully mature steroid hormone receptor.

Hox gene expression in the embryonic genital system of the sea turtle Lepidochelys olivacea (Eschscholt, 1829), a species with temperature-dependent sex determination.

Hox genes are conserved transcription factors which regulate embryonic morphogenesis and differentiation. For the first time, we examined the quantitative and spatial expression of two Hox 5' genes, HoxD11 and HoxA13, in the developing genital system of the olive ridley Lepidochelys olivacea, a species with temperature-dependent sex determination. Quantitative and spatial expression patterns of both genes suggest a role in the female pathway rather than the male pathway. For instance, both genes, especially HoxA13, were expressed in the undifferentiated gonad during the thermosensitive period at a female promoting temperature, and downregulated in the differentiated gonad. By contrast, expression of both genes was low in gonads incubated at a male promoting temperature and did not change significantly in the differentiated gonad. Furthermore, we found high expression levels of HoxA13 in the paramesonephric duct at the male promoting temperature but not at the female promoting temperature, suggesting a role for this Hox gene in the partial regression of the Müllerian duct in males.

Novel monoclonal antibodies against Pdx1 reveal feedback regulation of Pdx1 protein levels.

The aim of this study was to characterize two monoclonal antibodies (F6A11 and F109-D12) generated against Pdx1 (pancreatic and duodenal homeobox-1), a homeodomain transcription factor, which is critical for pancreas formation as well as for normal pancreatic beta cell function. For production of monoclonal antibodies, we immunized Robertsonian POSF (RBF)mice with a GST-Pdx1 fusion protein containing a 68-amino acid C-terminal fragment of rat Pdx1. These monoclonal antibodies detect Pdx1 by western blotting and allow immunohistochemical detection of Pdx1 in both mouse and rat tissue. F6A11 and F109-D12 produce IHC staining patterns indistinguishable from that obtained with highly specific polyclonal Pdx1 antisera raised in rabbits and goats, when applied to embryonic or adult mouse pancreatic tissue. In contrast to previously generated polyclonal anti-Pdx1 antisera, we also demonstrate that F6A11 works for intracellular fluorescence activated cell sorting (FACS) staining of Pdx1. By using F6A11, we characterize the induction of Pdx1 in the Doxycycline (DOX) inducible insulinoma cell line INSralphabeta-Pdx1 and follow the reduction of Pdx1 after removing Dox. Finally, we show that induction of exogenous Pdx1 leads to a reduction in endogenous Pdx1 levels, which suggests that a negative feedback loop is involved in maintaining correct levels of Pdx1 in the cell.

Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris.

Pancreatic duodenal hemeobox-1 (PDX1) is essential for the development of the embryonic pancreas and plays a key role in pancreatic beta-cell differentiation, maturation, regeneration, and maintenance of normal pancreatic beta-cell insulin-producing function. Purified recombinant PDX1 (rPDX1) may be a useful tool for many research and clinical applications, however, using the Escherichia coli expression system has several drawbacks for producing quality PDX1 protein. To explore the yeast expression system for generating rPDX1 protein, the cDNA coding for the full-length human PDX1 gene was cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rPDX1 was secreted into the culture medium, had a molecular weight by SDS-PAGE of 50kDa, and was glycosylated. The predicted size of the mature unmodified PDX1 polypeptide is 31kDa, suggesting that eukaryotic post-translational modifications are the result of the increased molecular weight. The recombinant protein was purified to greater than 95% purity using a combined ammonium sulfate precipitation with heparin-agarose chromatography. Finally, 120mug of the protein was obtained in high purity from 1L of the culture supernatant. Bioactivity of the rPDX1 was confirmed by the ability to penetrate cell membranes and activation of an insulin-luciferase reporter gene. Our results suggest that the P. pastoris expression system can be used to produce a fully functional human rPDX1 for both research and clinical application.

Recombinant expression of a novel human transcriptional repressor HMBOX1 and preparation of anti-HMBOX1 monoclonal antibody.

HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta (DE3) and induced by 1 mM IPTG for 4 h at 37 degrees Celsius. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni2+-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.

Biochemical and functional characterization of six SIX1 Branchio-oto-renal syndrome mutations.

Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by hearing loss, branchial arch defects, and renal anomalies. Recently, eight mutations in the SIX1 homeobox gene were discovered in BOR patients. To characterize the effect of SIX1 BOR mutations on the EYA-SIX1-DNA complex, we expressed and purified six of the eight mutants in Escherichia coli. We demonstrate that only the most N-terminal mutation in SIX1 (V17E) completely abolishes SIX1-EYA complex formation, whereas all of the other mutants are able to form a stable complex with EYA. We further show that only the V17E mutant fails to localize EYA to the nucleus and cannot be stabilized by EYA in the cell. The remaining five SIX1 mutants are instead all deficient in DNA binding. In contrast, V17E alone has a DNA binding affinity similar to that of wild type SIX1 in complex with the EYA co-factor. Finally, we show that all SIX1 BOR mutants are defective in transcriptional activation using luciferase reporter assays. Taken together, our experiments demonstrate that the SIX1 BOR mutations contribute to the pathology of the disease through at least two different mechanisms that involve: 1) abolishing the formation of the SIX1-EYA complex or 2) diminishing the ability of SIX1 to bind DNA. Furthermore, our data demonstrate for the first time that EYA: 1) requires the N-terminal region of the SIX1 Six domain for its interaction, 2) increases the level of the SIX1 protein within the cell, and 3) increases the DNA binding affinity of SIX1.

Purification of human transcription factors Nanog and Sox2, each in complex with Skp, an Escherichia coli periplasmic chaperone.

Nanog and Sox2 are key transcriptional factors involved in self-renewal and pluripotency of stem cells in human and other mammals. Nanog and Sox2 contain homeodomain (HD) and high-mobility group (HMG) DNA-binding domain, respectively, for targeting them to their regulatory regions and the other regions with transactivation function by providing sites for recruiting other transcriptional regulators. To gain insights in the biochemical and biophysical characteristics of the other regions of Nanog and Sox2, we have tried to overproduce and purify full length wild-type human Nanog and Sox2 expressed in Escherichia coli. Interestingly, we found that Nanog and Sox2 were individually stabilized by tight interaction with Skp, an E. coli periplasmic chaperone, thereby enabling stable over-expression and purification of Nanog and Sox2, each in complex with Skp. Purified Skp complexes of Nanog and Sox maintained DNA-binding activity toward its cognate DNA sequence. A similar approach may be applicable for some other mammalian proteins that are unstable or difficult to over-express in E. coli.

Molecular cloning and characterization of porcine homeodomain transcription factor Msx1.

We cloned a porcine ortholog of homeodomain transcription factor Msx1 from the porcine pituitary cDNA library. The amino acid sequence of Msx1 shows high conservation among mammalian species. RT-PCR for porcine fetal and postnatal pituitaries showed that Msx1 is already expressed at early fetal day 40, decreases to a low level before birth and then remarkably decreases after birth. On the other hand, Msx1 expression was observed in all pituitary-derived cell line tested, with most in a gonadotrope lineage LbetaT4. Transfection assay demonstrated that Msx1 markedly repressed the basal Cga and Fshb gene expression, while Lhb expression was affected slightly. Taken together, Msx1 may play a role in repressing gene expression in the fetal and postnatal periods.

Purification of caudal-related homeodomain transcription factor and its binding characterization.

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in the Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatography. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2 binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.

Expression and purification of human full-length N Oct-3, a transcription factor involved in melanoma growth.

This report describes the first purification procedure of the human full-length N Oct-3 protein in amounts suitable for structural studies and proteomic investigations. N Oct-3 is a transcription factor member of the POU protein family. It possesses a large N-terminal transactivation domain and a DNA-binding domain (DBD) which is composed of two subdomains, POUs and POUh, which are joined by a linker peptide. N Oct-3 is a master gene for central nervous system development but also for melanoma progression. Previous structural studies have all been performed using N Oct-3 DBD only. In this study, the full-length N Oct-3 protein was bacterially expressed and purified to homogeneity. The purified protein gave a single band at approximately 53 kDa on SDS-PAGE, while cDNA sequence analysis revealed a calculated molecular mass of 47 kDa confirmed by mass spectroscopy. Size-exclusion chromatography experiments indicated that in solution, full-length N Oct-3 was a monomer. Circular dichroïsm and intrinsic tryptophan fluorescence showed that full-length N Oct-3 was folded, with a significant alpha-helix content probably located in its DBD. Comparison with the purified N Oct-3 DBD demonstrated that, at least in vitro, the affinity of the protein for its DNA targets was similar. This suggests that the transactivation domain of N Oct-3 was not involved in N Oct-3 DNA interaction.