Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain.

Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.

Expression and purification of the human tumor suppressor protein RNASET2 in CHO-S cells.

Members of the T2 extracellular ribonucleases family have long been reported as stress response proteins, often involved in host defence, in many different taxonomic groups. In particular, the human RNASET2 protein (hRNASET2) has been reported as an extracellular tumor suppressor protein, endowed with the ability to act as an "alarmin" signalling molecule following its expression and secretion in the tumor microenvironment by cancer cells and the subsequent recruitment and activation of cells belonging to the host innate immune system. Many in vitro and in vivo assays have been recently reported in support of the oncosuppressive role of hRNASET2: most of them relied on genetically engineered cell lines and the use of recombinant proteins from non-mammalian sources. In order to ensure a human-like glycosylation pattern, here we report for the first time the expression of recombinant hRNASET2 in the CHO-S cell line. We established a simple one-step chromatographic purification procedure that resulted in the production of 5 mg of endotoxin-free hRNASET2 per liter of culture, with a >95% purity degree. hRNASET2 expressed in CHO-S cells displayed a high degree of glycosylation homogeneity and a secondary structure content in agreement with that determined from the crystal structure. Indeed, recombinant hRNASET2 was active at both enzymatic and functional level, as stated by a biological activity assay. The availability of a pure, homogeneous recombinant human RNASET2 would provide a key tool to better investigate its non cell-autonomous roles in the context of cancer development and growth.

A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts.

We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.

DMBT1 inhibition of Pseudomonas aeruginosa twitching motility involves its N-glycosylation and cannot be conferred by the Scavenger Receptor Cysteine-Rich bacteria-binding peptide domain.

The scavenging capacity of glycoprotein DMBT1 helps defend mucosal epithelia against microbes. DMBT1 binding to multiple bacterial species involves its conserved Scavenger Receptor Cysteine-Rich (SRCR) domains, localized to a 16-mer consensus sequence peptide, SRCRP2. Previously, we showed that DMBT1 bound Pseudomonas aeruginosa pili, and inhibited twitching motility, a pilus-mediated movement important for virulence. Here, we determined molecular characteristics required for twitching motility inhibition. Heat-denatured DMBT1 lost capacity to inhibit twitching motility and showed reduced pili binding (~40%). Size-exclusion chromatography of Lys-C-digested native DMBT1 showed that only high-Mw fractions retained activity, suggesting involvement of the N-terminal containing repeated SRCR domains with glycosylated SRCR-Interspersed Domains (SIDs). However, individual or pooled consensus sequence peptides (SRCRPs 1 to 7) showed no activity and did not bind P. aeruginosa pili; nor did recombinant DMBT1 (aa 1-220) or another SRCR-rich glycoprotein, CD163. Enzymatic de-N-glycosylation of DMBT1, but not de-O-glycosylation, reduced its capacity to inhibit twitching motility (~57%), without reducing pili binding. Therefore, DMBT1 inhibition of P. aeruginosa twitching motility involves its N-glycosylation, its pili-binding capacity is insufficient, and it cannot be conferred by the SRCR bacteria-binding peptide domain, either alone or mixed with other unlinked SRCRPs, suggesting an additional mechanism for DMBT1-mediated mucosal defense.

Stromal expression of JNK1 and VDR is associated with the prognosis of esophageal squamous cell carcinoma

Purpose: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide, and its outcome is poor. The purpose of this study was to determine the association between JNK1 and vitamin D receptor (VDR) expression and the prognosis of ESCC. Methods: Immunohistochemical staining was conducted on ESCC tissue microarrays (362 pairs of ESCC and normal esophagus tissues). The epithelial and stromal expression levels of c-jun NH2-terminal kinase 1 (JNK1) and VDR were scored and correlated with the ESCC characteristics. Laser-capture-based quantitative RT-PCR was performed on ESCC tissues. The effects of JNK1 and VDR on ESCC cell proliferation and migration were analyzed in vitro by transient transfection, and protein changes were evaluated by immunoblotting. Results: Both JNK1 and VDR were reduced in ESCC epithelial cells in comparison with the normal esophagus, but the expression of JNK1 and VDR in ESCC stromal tissues, not epithelial cells, was strongly associated with the survival time of ESCC patients. Functional studies showed that increased JNK1 suppressed cancer cell proliferation, mobility, and migration, which were linked to the alterations of VDR and metastasis-associated proteins. Conclusion: JNK1 and VDR act as tumor suppressors, and their stromal expression levels are associated with prognosis in esophageal squamous cell carcinoma

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Expression and purification of a rapidly degraded protein, TMEM8B-a, in mammalian cell line.

TMEM8B-a protein is the longer, predominant isoform of the TMEM8B gene product, which is a tumor metastasis suppressor in nasopharyngeal carcinoma (NPC) and lung cancer. TMEM8B-a is rapidly degraded via the proteasome pathway mediated by ezrin in many NPC and lung cancer cell lines, but TMEM8B-a is not ubiquitinated. In this study, we report the recombinant production of full-length modified TMEM8B-a in mammalian cells. We used the PiggyBac transposon system to efficiently generate normal and lung cancer cell lines with stable TMEM8B-a protein expression. 293FT cells were the best host cell line to express TMEM8B-a protein. Then, we treated the stable 293FT cell lines with various small-molecule inhibitors and demonstrated that treatment with MG-132 and bortezomib, which target the proteasome and disrupt its function, could prevent TMEM8B-a degradation and induce protein expression in 293FT cells. Finally, we utilized the combination of Twin-Strep-tag and Strep-Tactin XT resin to successfully purify the TMEM8B-a protein. The final yield was estimated to be approximately 10-20 µg of the purified TMEM8B-a per 3.0 × 108 293FT cells.

Expression and purification of TAT-NDRG2 recombinant protein and evaluation of its anti-proliferative effect on LNCaP cell line.

N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti-proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E. coli-BL21(DE3). TAT-NDRG2 and NDRG2 proteins were expressed in the bacteria, purified using affinity chromatography and verified using western blotting. The effects of TAT-NDRG2 and NDRG2 protein treatment on LNCaP cells proliferation and apoptosis were evaluated using MTT assay and AnnexinV, 7-AAD flow cytometry assay, respectively. Western blot analysis confirmed the expression and purification of TAT-NDRG2 and NDRG2 proteins. Treatment of LNCaP cells with TAT-NDRG2 protein increased cell death and induced apoptosis significantly (P < 0.05) compared to control and NDRG2 protein-treated cells. These results suggest that TAT-NDRG2 protein can be considered as a therapeutic modality for the treatment of tumors.

Screening of Tumor Suppressor Genes in Metastatic Colorectal Cancer.

Most tumor suppressor genes are commonly inactivated in the development of colorectal cancer (CRC). The activation of tumor suppressor genes may be beneficial to suppress the development and metastasis of CRC. This study analyzed genes expression and methylation levels in different stages of CRC. Genes with downregulated mRNA expression and upregulated methylation level in advanced CRC were screened as the potential tumor suppressor genes. After comparing the methylation level of screened genes, we found that MBD1 gene had downregulated mRNA expression and upregulated methylation levels in advanced CRC and continuously upregulated methylation level in the progression of CRC. Enrichment analysis revealed that genes expression in accordance with the elevated expression of MBD1 mainly located on chromosomes 17p13 and 17p12 and 8 tumor suppressor genes located on chromosome 17p13. Further enrichment analysis of transcription factor binding site identified that SP1 binding site had higher enrichment and could bind with MBD1. In conclusion, MBD1 may be a tumor suppressor gene in advanced CRC and affect the development and metastasis of CRC by regulating 8 tumor suppressor genes through binding with SP1.

CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag.

There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis.

Voltage-sensitive rhodol with enhanced two-photon brightness.

We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using two-photon (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue.

A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle.

Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus.

[Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation.

New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris.

Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called "tumor antagonizing genes," whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression.

Autophagy-independent functions of UVRAG are essential for peripheral naive T-cell homeostasis.

UV radiation resistance-associated gene (UVRAG) encodes a tumor suppressor with putative roles in autophagy, endocytic trafficking, and DNA damage repair but its in vivo role in T cells is unknown. Because conditional homozygous deletion of Uvrag in mice results in early embryonic lethality, we generated T-cell-specific UVRAG-deficient mice that lacked UVRAG expression specifically in T cells. This loss of UVRAG led to defects in peripheral homeostasis that could not be explained by the increased sensitivity to cell death and impaired proliferation observed for other autophagy-related gene knockout mice. Instead, UVRAG-deficient T-cells exhibited normal mitochondrial clearance and activation-induced autophagy, suggesting that UVRAG has an autophagy-independent role that is critical for peripheral naive T-cell homeostatic proliferation. In vivo, T-cell-specific loss of UVRAG dampened CD8(+) T-cell responses to LCMV infection in mice, delayed viral clearance, and impaired memory T-cell generation. Our data provide novel insights into the control of autophagy in T cells and identify UVRAG as a new regulator of naïve peripheral T-cell homeostasis.

TAT-RhoGDI2, a novel tumor metastasis suppressor fusion protein: expression, purification and functional evaluation.

Rho GDP dissociation inhibitor 2 (RhoGDI2) was identified as a functional metastasis suppressor in human bladder cancer, suggesting that increasing the RhoGDI2 level may represent a promising therapeutic strategy. It has been shown that the transactivator of transcription (TAT) protein from HIV-1 is able to efficiently deliver various biological molecules into several cell types. In this study, TAT peptide was fused with the N-terminus of RhoGDI2, and the resulting TAT-RhoGDI2 fragment was inserted into the pGEX-6p-1 plasmid and expressed as a glutathione S-transferase (GST)/TAT-RhoGDI2 fusion protein in Escherichia coli BL21(DE3) cells. A two-step purification strategy involving glutathione sepharose chromatography and PreScission protease cleavage was developed to purify TAT-RhoGDI2; subsequently, the identification of the involved macromolecules was achieved by Western blot. The final product, TAT-RhoGDI2, was obtained at a concentration of 112 mg/L. This is the first report on the efficient production of bioactive TAT-RhoGDI2 through a gene-engineering approach in E. coli. Using flow cytometry, we found that the TAT-RhoGDI2 fusion proteins could penetrate into bladder cancer cells with an extremely high efficiency. In vitro scratch and transwell assay and the migration/invasion behavior of UMUC3 cells were strongly reduced by the treatment with TAT-RhoGDI2. These studies support the use of the TAT-RhoGDI2 protein in tumor metastasis therapy.

A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies.

The L10 ribosomal protein (RPL10) plays a role in the binding of the 60 S and 40 S ribosomal subunits and in mRNA translation. The evidence indicates that RPL10 also has multiple extra-ribosomal functions, including tumor suppression. Recently, the presence of RPL10 in prostate and ovarian cancers was evaluated, and it was demonstrated to be associated with autistic disorders and premature ovarian failure. In the present work, we successfully cloned and expressed full-length human RPL10 (hRPL10) protein and isolated inclusion bodies containing this protein that had formed under mild growth conditions. The culture produced 376mg of hRPL10 protein per liter of induced bacterial culture, of which 102.4mg was present in the soluble fraction, and 25.6mg was recovered at approximately 94% purity. These results were obtained using a two-step process of non-denaturing protein extraction from pelleted inclusion bodies. We studied the characteristics of this protein using circular dichroism spectroscopy and by monitoring the changes induced by the presence or absence of zinc ions using fluorescence spectrometry. The results demonstrated that the protein obtained using these non-conventional methods retained its secondary and tertiary structure. The conformational changes induced by the incorporation of zinc suggested that this protein could interact with Jun or the SH3 domain of c-yes. The results suggested that the strategy used to obtain hRPL10 is simple and could be applied to obtaining other proteins that are susceptible to degradation.

Interaction of Grb2 SH3 domain with UVRAG in an Alzheimer's disease-like scenario.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein which participates in trafficking pathways alongside its role in signaling. Proteins important for actin remodeling and cellular compartmentalization contain SRC Homology 3 (SH3) binding motifs that interact with Grb2. While studying the Grb2-amyloid precursor protein (APP) intracellular domain (AICD) interaction in Alzheimer's disease cell line models, it was seen that Grb2 colocalized to compartments that mature into autophagosomes. The entrapping of AICD in the Grb2 vesicles and its clearance via autophagosomes was a survival contrivance on the part of the cell. Here, we report that Grb2, when in excess, interacts with ultraviolet radiation resistance-associated gene protein (UVRAG) under excess conditions of AICD-Grb2 or Grb2. The N-terminal SH3 domain of Grb2 specifically interacts with UVRAG, unlike the C-terminal SH3 domain. This interaction helps to understand the role of Grb2 in the autophagic maturation of vesicles.

Glomerular autoimmune multicomponents of human lupus nephritis in vivo: α-enolase and annexin AI.

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum.

Direct electrochemical DNA detection originated from the self-redox signal of sulfonated polyaniline enhanced by graphene oxide in neutral solution.

In this paper, a type of direct DNA impedance detection using the self-redox signal change of sulfonated polyaniline (SPAN) enhanced by graphene oxide (GNO) was reported, here SPAN is a copolymer obtained from aniline and m-aminobenzenesulfonic acid. The resulting nanocomposite was characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The π-π planar structure of GNO and the carboxyl groups on the surface of GNO ensured it could act as an excellent substrate for adsorption and polymerization of aniline monomer. Because of the existence of GNO, the electrochemical activities of SPAN were enhanced obviously. Because of abundant sulfonic acid groups, the resulting nanocomposite showed obvious self-redox signal even at physiological pH, which is beneficial for biosensing field. DNA probes with amine groups could be covalently attached to the modified electrode surface through the acyl chloride cross-linking reaction of sulfonic groups and amines. When the flexible probe DNA was successfully grafted, the electrode was coated and electron transfer between electrode and buffer was restrained. Thus, the inner impedance value of SPAN (rather than using outer classic EIS probe, [Fe(CN)6](3-/4-)) increased significantly. After hybridization, the rigid helix opened the electron channel, which induced impedance value decreased dramatically. As an initial application of this system, the PML/RARA fusion gene sequence formed from promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) was successfully detected.

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.