kDa Fusion Protein Research Articles

Highly sensitive and quantitative HiBiT-tagged Nipah virus-like particles: A platform for rapid antibody neutralization studies

Biocontainment regulations restrict the research on NiV to BSL-4 laboratories, thus limiting the mechanistic studies related to viral entry and allied pathogenesis. Understanding the precise process of viral-particle production and host cell entry is critical for designing targeted therapies or particle-based vaccines. In this study, we have synthesized HiBiT-tagged-NiV-VLPs to ease in-vitro BSL-2 particle handling. We propose a simple yet effective approach of generating substantial amount of HiBiT-tagged NiV-VLPs in vitro by co-expressing viral structural proteins in HEK293T cells. Though homologous to parent virus, the incapacitated replication potential facilitates a BSL-2 handling of these particles. The inclusion of a highly sensitive HiBiT tag on these VLPs allows for a quick detection of viral binding and entry, as well as in assessing the efficiency of neutralizing antibodies in vitro using the NanoBiT technology. The HiBiT-tag binds in high affinity with LgBiT (Large BiT an 18 kDa fusion protein and complementary subunit of HiBiT peptide), and the resultant complex elicits high intensity luminescence in the presence of substrate. The VLPs produced were morphologically and functionally identical to the native virus, and the HiBiT-tag permitted their quick application in viral binding, entry, and antibody neutralization assays.“Thus, we report a simple setting for generating HiBiT-NiV VLPs which can be utilized in a BSL-2 laboratory, to concurrently quantify features of NiV assembly, binding and entry. This also offers an alternate-safe and effective platform for viral based antibody neutralization assays in vitro”.

Concurrent BCR::-ABL1 and CBFB Rearrangement in De Novo Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is known for its genetic heterogeneity. Several recurrent genetic variants have been characterized including AML-defining chromosomal rearrangements involving the core binding factor (CBF), such as inv(16)/t(16;16)/ CBFB:: MYH11. 1 Less common is a subset of AML characterized by the Philadelphia chromosome (Ph+), generating a BCR::ABL1 fusion protein. 2 The Ph+ AML subtype is associated with poor prognosis and treatment resistance. 3,4 Co-occurrence of CBFB rearrangement and BCR::ABL1 fusion is rare, representing a subgroup with unique clinical and molecular features. To date, there have been less than 30 cases reported in the literature. 5-8 Case Description A 62-year-old male presented to hospital with headaches, visual changes, weakness, and a nodular rash on his back, with leukocytosis, thrombocytopenia, and 42.5% circulating blasts. A bone marrow (BM) aspirate and biopsy was diagnostic of AML with 56% myeloblasts. RT-PCR was positive for an e19a2 BCR::ABL1 fusion transcript coding for p230 kDa fusion protein (p230) and CBFB: :MYH11 fusion gene transcript. Cytogenetics identified a complex karyotype with an inv(16) and a Ph chromosome (46,XY,t(1;14)(p32;q24),t(9;22)(q34;q11.2),inv(16)(p13.1q22)[10]), consistent with concurrent BCR::ABL1 and CBFB::MYH11 rearrangements within the same clonal population of cells. Treatment consisted of hydroxyurea for cytoreduction, 7+3 induction, one cycle of low dose cytarabine consolidation, and oral azacytidine maintenance, in combination with dasatinib. He did not receive Gemtuzumab ozogamicin (GO) given patient preference and comorbidities. Post-induction course was complicated by febrile neutropenia due to Enterococcus Faceium bacteremia, pre-renal acute kidney injury, and dasatinib-related complications including rash and transaminitis. These issues resolved by holding dasatinib for 15 days. Complete morphologic remission (CR) was obtained by Day 33 post-induction and no fusion gene transcripts were detectable by nested RT-PCR from BM aspirate on day 49 post-induction, nor day 76 post-consolidation The patient was ineligible for an allogeneic stem cell transplant given his poor performance status. At present, the patient remains alive and well, continuing on maintenance with oral azacytidine and dasatinib. Discussion We present the only case, to our knowledge, of a concurrent BCR::ABL1 p230 isoform and CBFB::MYH11 with de novo AML. Based on our literature search, 26 cases have been reported. BCR::ABL1 and CBFB::MYHII rearrangements in de novo AML potentially confers an intermediate to favorable prognosis with outcomes comparable to those of CBF-AML. Studies suggest that this entity may be more akin to AML with inv(16) rather than CML-BP with inv(16), thereby retaining favorable prognosis. 5,9-11 There are no formal guidelines for management of de novo AML with concurrent BCR::ABL1 and CBFB rearrangements. Traditional chemotherapy treatment strategies for Ph+ AML are often ineffective in these cases. The presence of BCR::ABL1 fusion transcript raises the possibility of utilizing TKIs targeting the BCR::ABL1 kinase domain. In our case, the patient achieved rapid CR with induction chemotherapy and dasatinib, but experienced multiple complications, precluding further intensive chemotherapy and allogeneic stem cell transplantation. This case raises a number of questions, including the optimal timing, combination, and duration of TKI therapy in the context of dual rearrangement de novo AML. This patient subgroup may benefit from intensive chemotherapy regimens and TKIs. Prospective monitoring of these cases is warranted to assess outcomes in comparison to Ph+ AML without coinciding CBFB rearrangement. This case report underscores the importance of comprehensive genetic profiling in Ph+ AML to determine the genetic landscape and individualize therapeutic strategies.

Expression and copper binding studies of a Plasmodium falciparum protein with Cox19 copper binding motifs

Copper can exist in an oxidized and a reduced form, which enables the metal to play essential roles as a catalytic co-factor in redox reactions in many organisms. Copper confers redox activity to the terminal electron transport chain cytochrome c oxidase protein. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11 in association with Cox19. When copper is chelated in growth medium, Plasmodium falciparum parasite development in infected red blood cells is inhibited and excess copper is toxic for the parasite. The gene of a 26 kDa Plasmodium falciparum PfCox19 protein with two Cx9C Cox19 copper binding motifs, was cloned and expressed as a 66 kDa fusion protein with maltose binding protein and affinity purified (rMBP-PfCox19). rMBP-PfCox19 bound copper measured by: a bicinchoninic acid release assay; an in vivo bacterial host growth inhibition assay; ascorbate oxidation inhibition and differential scanning fluorimetry. The native protein was detected by antibodies raised against rMBP-PfCox19. PfCox19 binds copper and is predicted to associate with PfCox11 in the insertion of copper into the CuB site of Plasmodium cytochrome c oxidase. Characterisation of the proteins involved in Plasmodium spp. copper metabolism will help us understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.

A candidate subunit vaccine induces protective immunity against Mycobacterium avium subspecies paratuberculosis in mice

Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis (PTB), which is a granulomatous enteritis in ruminants that threatens the dairy industry’s healthy development and public health safety worldwide. Because the commercial inactivated vaccines are not completely protective and interfere with bovine tuberculosis diagnostics, we tested four fusion proteins, namely 66NC, 66CN, 90NC, and 90CN, which were constructed with MAP3527, Ag85B, and Hsp70 of MAP in different tandem combinations. Notably, 66NC, which encodes a 66 kDa fusion protein that combines in linear order MAP3527N40–232, Ag85B41–330, and MAP3527C231–361, induced a powerful and specific IFN-γ response. Immunization of C57BL/6 mice with the 66NC fusion protein formulated in Montanide ISA 61 VG adjuvant generated robust Th1, Th2, and Th17 type immune responses and strong antibody responses. The 66NC vaccine protected C57BL/6 mice against virulent MAP K-10 infection. This resulted in a reduction of bacterial load and improvement of pathological damage in the liver and intestine, in addition to a reduction of body weight loss; significantly better protection than the reported 74 F vaccine was also induced. Furthermore, vaccine efficacy correlated with the levels of IFN-γ-, TNF-α-, and IL-17A-secreting antigen-specific CD4+ and CD8+ T lymphocytes as well as with serum IFN-γ and TNF-α levels after vaccination. These results demonstrate that recombinant protein 66NC is an efficient candidate for further development into a protective vaccine in terms of inducing specific protection against MAP.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

Biểu hiện gen fliC cải biến của Salmonella Dublin và tinh sạch flagellin tái tổ hợp

Flagellin is a potent trigger of innate immune responses that enhance adaptive immune responses. Flagellin has intrinsic adjuvant activity mediated through toll-like receptor (TLR 5) and is an attractive candidate for highly effective vaccine adjuvant. The N-terminal D0-D1 domain of flagellin is required for TLR5 recognition. In the present study, fliC gene from S. Dublin expressed in E. coli BL21 in the fusion form with SUMO protein. The 48 kDa fusion protein of SUMO-flagellin was synthesised in insoluble inclusion body. The overall yield of expressed SUMO - flagellin was 0.6 g/L. The recombinant protein solubilized in 2 M urea then was purified by using Ni-NTA column chromatography with purity about 95.4%. Flagellin will be cleaved from fusion protein and used to develop as a radiation countermeasure.

In silico designing and expression of novel recombinant construct containing the variable part of CD44 extracellular domain for prediagnostic breast cancer.

CD44, as a tumor-associated marker, can be used to detect stem cells in breast cancer. While CD44 is expressed in normal epithelial cells, carcinoma cells overexpress CD44. In the current study, we designed a recombinant protein that included the variable component of the CD44 (CD44v) extracellular domain to apply in clinical diagnosis of breast cancer. A total of 100 CD44v amino-acid residues were determined, and the structure was examined using bioinformatics tools. The construct was inserted into the PET28a vector and transformed in E. coli BL21(DE3). A nearly 12 kDa fusion protein was obtained by Ni-NTA affinity metal chromatography. Recombinant CD44v was examined by Western blotting, ELISA, and immunohistochemistry (IHC) assays. The findings revealed that the structure of rCD44v was stable, and its antigenic domain was exposed. The recombinant CD44v was confirmed by western blotting, and the presence of antibodies against recombinant CD44v protein in the patient's serum was detected by the ELISA. Our data demonstrated a link between CD44v serum levels and the prevalence of breast cancer. Assessments of antiCD44v antibodies with rCD44v could be a useful tool for identifying breast cancer in its early stages, which can lead to better outcomes.

Development of polyclonal antibodies using bacterially expressed recombinant coat protein for the detection of Onion yellow dwarf virus (OYDV) and identification of virus free onion genotypes

Onion yellow dwarf virus (OYDV) belonging to the genus Potyvirus, family Potyviridae, is one of the widely distributed viruses of Allium species worldwide. It causes dwarfing, yellow striping, crinkling and flaccidity of the leaves of onion and garlic. To see the occurrence and incidence of OYDV on Allium crop, an attempt was made to develop antibody based diagnostic assay which would be useful for routine indexing and screening of the germplasm. The total RNA was isolated from the symptomatic leaves of onion and the gene encoding coat protein (CP) was cloned. The nucleotide sequencing analysis of the cloned RT-PCR product revealed ~ 774bp amplicon (OYDV CP) and it was further cloned in pET-28a ( +) expression vector which yielded ~ 30kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was primarily checked on SDS-PAGE and further confirmed by Western blot. The His6BP-OYDV-CP was obtained in soluble state after purification and was used to immunize New Zealand white rabbit for the production of polyclonal antibody (PAb). The produced PAb against the purified fusion protein successfully detected OYDV from onion and garlic samples at 1:2000 dilutions in indirect-enzyme linked immunosorbent assay (DAC-ELISA). Thus, this study presents first report that Histidine tag (His6BP) fusion OYDV-CP based antibody production and its successful application in identification of virus free onion and garlic genotypes.

Cloning and characterization of the Bambusa oldhamii BoMDH-encoded malate dehydrogenase

Malate dehydrogenase (MDH), which is ubiquitously occurred in nature, catalyzes the interconversion of malate and oxaloacetate. Higher plants contain multiple forms of MDH that differ in coenzyme specificity, subcellular localization and physiological function. A putative Bambusa oldhamii BoMDH cDNA was screened with the specific probe from the bamboo cDNA library. Sequence alignment shows that there's a high homology between the deduced amino acid sequence of BoMDH and MDH protein in Oryza sativa glyoxysome (92%). A 57 kDa fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the recombinant protein. The molecular mass of recombinant BoMDH was estimated to be 120 kDa, and the subunit form was 57 kDa by denatured SDS-PAGE, indicating that BoMDH presents as a homodimer. The optimum temperature and pH for BoMDH activity were 40 °C and 9.5, respectively. The Km values of BoMDH for malate and NAD+ were 5.2 mM and 0.52 mM. The kcat/Km values of BoMDH for malate and NAD+ were 163 min−1 mM−1 and 3060 min−1 mM−1.

Expression and purification of thioredoxin-his6-ZmDREB2.7 fusion protein in Escherichia coli for raising antibodies

Dehydration-responsive element-binding (DREB) proteins play a critical role in the plant’s drought-tolerance mechanism despite their presence in minor amounts in the cell. In this study, a maize-derived transcription factor protein, ZmDREB2.7, was overexpressed in the Escherichia coli strain Rosetta 1. The interested gene conjugating with the thioredoxin gene (TrxA) and his6 tag in the pET-32a vector encoded a 55.7 kDa fusion protein. The optimum condition for inducing the thioredoxin-his6-ZmDREB2.7 expression was five hours of induction with 0.05 mM IPTG at 300C. The Tris-HCl 20 mM pH 8.0 lysis buffer was harnessed to extract the recombinant protein for the purification process. Using the immobilized-metal affinity chromatography column, the recombinant protein was purified and then injected into rabbits. The antisera containing polyclonal antibodies (pAbs) could specifically recognize the ZmDREB2.7 fusion protein. This study represents updated data on the bacterial expression of the recombinant ZmDREB2.7 protein and the production of anti-ZmDREB2.7 pAbs.

Development of immunodiagnostics for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.

ZFP226 is a novel artificial transcription factor for selective activation of tumor suppressor KIBRA

KIBRA has been suggested as a key regulator of the hippo pathway, regulating organ size, cell contact inhibition as well as tissue regeneration and tumorigenesis. Recently, alterations of KIBRA expression caused by promotor methylation have been reported for several types of cancer. Our current study aimed to design an artificial transcription factor capable of re-activating expression of the tumor suppressor KIBRA and the hippo pathway. We engineered a new gene named ‘ZFP226′ encoding for a ~23 kDa fusion protein. ZFP226 belongs to the Cys2-His2 zinc finger type and recognizes a nine base-pair DNA sequence 5′-GGC-GGC-GGC-3′ in the KIBRA core promoter P1a. ZFP226 showed nuclear localization in human immortalized kidney epithelial cells and activated the KIBRA core promoter (p < 0.001) resulting in significantly increased KIBRA mRNA and protein levels (p < 0.001). Furthermore, ZFP226 led to activation of hippo signaling marked by elevated YAP and LATS phosphorylation. In Annexin V flow cytometry assays ZFP226 overexpression showed strong pro-apoptotic capacity on MCF-7 breast cancer cells (p < 0.01 early-, p < 0.001 late-apoptotic cells). We conclude that the artificial transcription factor ZFP226 can be used for target KIBRA and hippo pathway activation. This novel molecule may represent a molecular tool for the development of future applications in cancer treatment.

Molecular characterization of duck enteritis virus UL41 protein

BackgroundDuck enteritis virus (DEV) belongs to the subfamily Alphaherpesvirinae, and information on the DEV UL41 gene is limited.MethodsThe DEV UL41 gene was cloned into the pET32a(+) vector and expressed in a prokaryotic expression system. Antiserum was raised against a bacterially expressed UL41-His fusion protein for further experiments. Transcription was quantified and UL41 protein expression levels were determined in DEV-infected cells at different time points by RT-qPCR and western blotting, respectively. DEV virions were purified by sucrose gradient centrifugation and analyzed by mass spectrometry to identify protein content. We confirmed the DEV UL41 gene kinetic class using a pharmacological test. IFA was used to analyze the intracellular localization of pUL41.ResultsThe recombinant expression plasmid, pET-32a(+)-UL41, which highly expresses a 76.0 kDa fusion protein, was constructed and expressed in E. coli BL21 (DE3) after induction with 0.2 mM IPTG at 30 °C for 10 h, generating a specific mouse anti-UL41 protein polyclonal antibody. RT-qPCR and western blot analyses revealed that the UL41 transcript number peaked at 36 hpi, and peak protein expression occurred at 48 hpi. The pharmacological test showed that UL41 was a γ2 gene. Mass spectrometry analysis showed that pUL41 was a virion component. IFA results revealed that pUL41 was localized throughout DEV-infected cells but only localized to the cytoplasm of transfected cells. DEV pUL47 translocated pUL41 to the nuclei of DEF cells; this translocation was dependent on predicted pUL47 NLS signals (40–50 aa and 768–777 aa).ConclusionsDEV UL41 is a γ2 gene that encodes a virion structural protein, pUL41 localizes throughout DEV-infected cells but only localizes to the cytoplasm of transfected cells. pUL41 cannot autonomously localize to the nucleus, as this nuclear localization is dependent on predicted DEV pUL47 NLS signals (40–50 aa and 768–777 aa).

Molecular characterization and functional analysis of the hepcidin gene from roughskin sculpin (Trachidermus fasciatus)

Hepcidin is a kind of cysteine-rich antimicrobial peptide that plays a vital role in host innate immune activity and iron regulation. Here, we report the molecular characterization and functional analysis of a novel hamp1 hepcidin isoforms Tf-Hep from roughskin sculpin, Trachidermus fasciatus. A cDNA fragment of 988 bp with an ORF of 273 bp was obtained. The coding sequence encodes for a signal peptide of 24 amino acids coupled with a prodomain of 40 amino acids and a mature peptide of 26 amino acids. Tissue distribution analysis indicated that Tf-Hep was most abundant in the liver. It could be significantly induced post lipopolysaccharide (LPS) challenge and heavy metal exposure. The mature peptide was expressed as a 6.061 kDa fusion protein in Pichia pastoris GS115. The active purified recombinant protein (rTf-Hep) exhibited a wide spectrum of potent antimicrobial activity in vitro against 4 Gram-negative bacteria Escherichia coli, Vibrio Anguillarum, Klebsiella pneumoniae, and Pseudomonas aeruginosa and 4 Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus megaterium with minimum inhibitory concentrations (MICs) of 5–80 μg/ml (0.825–13.2 μM). It also displayed high affinity to polysaccharides on bacteria surface including LPS, lipoteichoic acid (LTA) and peptidoglycan (PGN). We further revealed that rTf-hep was capable of agglutinating 6 of the 8 bacteria. All these results suggest that rTf-hep may be both an antibacterial effector and a pattern recognition molecule in fish immune defense. The in vivo bacterial treatment results demonstrated that rTf-Hep could significantly improve the survival rate of fish infected with V. anguillarum. Taken together, these data indicate an important role for Tf-hep in the innate immunity of Trachidermus fasciatus and suggest its potential application in aquaculture for increasing fish resistance to disease.

Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag

The molecular structure of acetylcholinesterase (AChE) attracts interest because of its versatility and significant role in the cholinergic system. The main purpose of the present study was to clone a full-length cDNA sequence of human brain acetylcholinesterase (hAChE) into pET SUMO vector and express it successfully. The integrity of the constructed plasmid was confirmed by cross PCR. This recombinant construct was expressed in Escherichia coli BL21 (DE-3). In this work, we produced hexahistidine (6xHis) tagged fusion protein by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction and purified using nickel (Ni2+) affinity chromatography. Using anti-His antibody, we detected ~90 kDa fusion protein. The expression of the hAChE gene in a microbial host resulted in good biological activity. Using the Ellman method, the recombinant AChE exhibited activity with optima at pH 9.0 glycine-NaOH buffer and room temperature. Kinetic parameters, KM and Vmax, were determined as 0.63 and 0.69, respectively.

Existence of a weak cytotoxic CD4+ T cell population in mice infected with ectromelia virus

This study set to delineate MHC class II immunogenic peptides encoded in proteins expressed by A33R, 14 kDa fusion protein and p42 genes of ectromelia virus (ECTV) Moscow strain (ECTV-Mos), a virus related to variola virus (Variola vera virus) responsible for smallpox in humans. A search for a safe and efficacious vaccine against poxviruses is still required mostly because of the emerging nature of certain viruses among poxviruses. In silico prediction of peptides from the 3 protein sequences revealed 6 potential candidates. Investigations included assessment of the peptide’s ability to bind to MHC class II molecules on antigen-presenting cells and to induce the proliferation, cytokine synthesis and cytotoxicity of CD4+ T cells originating from mice previously infected with ECTV-Mos. The results show that peptide ENHAETLRAAMISLA (Pep3) predicted from the protein sequence 14 kDa fusion protein induced significant proliferation, cytokine synthesis and cytotoxicity. Also Pep3 was able to bind strongly to MHC class II molecules on A20 cells. These results suggest that a small population of CD4+ T cells play a protective role dependent on cytotoxicity and possibly complement the CD8+ T cells population in this regard.

NPM1-TYK2 Fusion Is an Oncogene and a Novel HSP90-Client Protein

Cutaneous CD30-positive lymphoproliferative disorders (LPDs), the second most common type of cutaneous T-cell lymphoma include a clinicopathologic spectrum of benign lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large-cell lymphoma (ALCL). NPM1-TYK2 is the first identified tyrosine kinase chromosomal translocation in CD30-positive LPDs. Understanding the deregulated functions resulting from new chromosomal translocations in these neoplasms is essential to discover novel therapeutic approaches. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Tyrosine kinase 2 (TYK2) is a non-receptor tyrosine kinase that belongs to the family of Janus Kinases (JAKs). NPM1-TYK2 is an 81 kDa fusion protein comprising of NPM1 (1-257 amino acids) and the kinase domain of TYK2 (726-1187 amino acids). Since NPM1-TYK2 is a recently discovered fusion gene, we conducted experiments to determine its oncogenic potential. Transduction of lentiviral particles bearing NPM1-TYK2 gene transformed the interleukin-3 (IL-3) dependent Ba/F3 cell line to IL-3 independent growth through constitutive activation of TYK2 kinase and downstream STAT signaling. We further evaluated the role of NPM1 as part of fusion protein in NPM1-TYK2 mediated oncogenicity. Transfection of NPM1-TYK2 deletion construct lacking NPM1 in HEK293 cells resulted in significant decrease in TYK2 kinase activity in the cells compared to full-length NPM1-TYK2 transfected cells. Currently, there are no effective small molecule inhibitors for NPM1/ TYK2 in the clinical trials. Hence, there is a clinical unmet need to develop novel, targeted therapies for NPM1-TYK2 driven lymphomas. As TYK2 is an established HSP90 client protein, the rationale for this study is to investigate the ability of HSP90 to regulate the stability and kinase activity of NPM1-TYK2 fusion protein. HSP90 is a master chaperone involved in the proper folding and maturation of a variety of oncogenic kinases and perturbing HSP90 function is emerging as a promising approach for cancer therapy. We used Myla cells (expressing endogenous NPM1-TYK2 fusion gene) to investigate the status of NPM1-TYK2 as a HSP90 client protein. Here we report that the treatment of cells with small molecule HSP90 inhibitor, 17-AAG promotes degradation of NPM1-TYK2 fusion protein resulting in downregulation of its oncogenically induced downstream STAT signaling pathways. Mechanistically, dephosphorylation of STAT1, 3, and 5 following 17-AAG treatment resulted in apoptotic cell death of the lymphoma cells. The results obtained using 17-AAG, were further corroborated using other HSP90 small molecule inhibitors AUY922 and PU-H71. Immunoprecipitation studies clearly demonstrate that 17-AAG treatment disrupts interaction between HSP90 and NPM1-TYK2 chimeric protein. Collectively, these findings provide an evidence for NPM1-TYK2 oncogenicity and therapeutic potential of HSP90 inhibitors for the treatment of a subset of cutaneous CD30-positive lymphoproliferative disorder patients expressing NPM1-TYK2 chimeric gene. DisclosuresGanguly:Onyx: Speakers Bureau; Seattle Genetics: Speakers Bureau; Janssen: Research Funding.

먹장어 Cathepsin L의 분자생물학적 클로닝, 발현 및 효소학적 특성 분석

Hagfish which belongs to the chordate contact cyclostomata, is important phylogenetic relationship between vertebrate and invertebrate. Cathepsins of the cysteine protease family have traditionally been thought to play a major role in intracellular protein degradation and turnover in lysosomes. In this study, Catepsin L was cloned from Inshore hagfish (Eptatretus burgeri), the cDNA encoding ORF of the Eptatretus burgeri Cathepsin L (EbCtL) is 978 bp. The cDNA encoding proEbCtL was expressed in Escherichia coli strain BL21(DE3) using the pGEX-4T-1 expression vector system. The recombinant proEbCtL protein was overexpressed as a approximately 55 kDa fusion protein. The overproduced soluble GST-fusion protein was then applied to glutathione-Sepharose 4B column chromatography; the sample harboring the fusion protein evidenced a high degree of purity when analyzed via SDS-PAGE and Western blot analysis. Its activity was quantied by cleaving the synthetic peptide Z-FR-AMC, Z-LLE-AMC, and Suc-AAF-AMC, and the optimal pH for the protease activity was 8, 9.5, and 9, respectively.

Development of recombinant vaccine candidate molecule against Shigella infection

Shigellosis is an acute bacillary diarrheal disease caused by the gram negative bacillus Shigella. The existence of multiple Shigella serotypes and their growing resistance to antibiotics stress the urgent need for the development of vaccine that is protective across all serotypes. Shigella’s IpaB antigen is involved in translocon pore formation, promotes bacterial invasion and induces apoptosis in macrophages. S. Typhi GroEL (Hsp 60) is the immunodominant antigen inducing both arms of immunity and has been explored as adjuvant in this study. The present study evaluates the immunogenicity and protective efficacy of recombinant IpaB domain-GroEL fusion protein in mice against lethal Shigella infection. The IpaB domain and GroEL genes were fused using overlap extension PCR and cloned in pRSETA expression vector. Fused gene was expressed in Escherichia coli BL-21 cells and the resulting 90 KDa fusion protein was purified by affinity chromatography. Intranasal (i.n.) immunization of mice with fusion protein increased the IgG and IgA antibody titers as compared to the group immunized with IpaB and GroEL and control PBS immunized group. Also IgG1 and IgG2a antibodies induced in fusion protein immunized mice were higher than co-immunized group. Significant increase in lymphocyte proliferation and cytokine levels (IFN-γ, IL-4 and IL-10), indicates induction of both Th1 and Th2 immune responses in both immunized groups. Immunization with fusion protein protected 90–95% of mice whereas 80–85% survivability was observed in co-immunized group against lethal challenge with S. flexneri, S. boydii and S. sonnei. Passive immunization conferred 60–70% protection in mice against all these Shigella species. Organ burden and histopathology studies also revealed significant decrease in lung infection as compared to the co-immunized group. Since IpaB is the conserved dominant molecule in all Shigella species, this study will lead to an ideal platform for the development of safe, efficacious and cost-effective recombinant vaccine against Shigella serotypes.

Protective antibody and cytokine responses in mice following immunization with recombinant beta-tubulin and subsequent Trypanosoma evansi challenge.

BackgroundTrypanosomosis or Surra, caused by the flagellated hemoprotozoan parasite Trypanosoma evansi, is a disease of economic importance through its wide prevalence in domestic livestock in tropical countries. In the absence of a protective vaccine, management of the disease relies on a few available chemotherapeutic agents. Although humoral immunity is the mainstay of resistance to T. evansi, the ability of the parasite to vary its immunodominant surface proteins to subvert the immune system has forced vaccine efforts to target a variety of invariant epitopes. Beta tubulin, an integral component of the trypanosome cytoskeleton, was therefore targeted using the recombinant form of the protein for immunization.MethodsThe 1329 bp coding sequence of beta tubulin gene was PCR amplified and cloned in pQE-TriSystem expression vector. Recombinant beta tubulin was heterologously expressed in Escherichia coli as a 46 KDa fusion protein and used for immunization of mice. The Ig response was studied by ELISA, whereas the cytokine response was measured using a cytometric bead-based assay quantified by flow cytometry.ResultImmunization with recombinant beta (β)-tubulin protein induced a beta-tubulin specific humoral immune response of predominantly IgG2a isotype. Lethal challenge with T. evansi blood-form trypomastigotes post-immunization elicited a robust anamnestic response. An abundance of IFN-γ further confirmed the Th-1 bias of the protective response. We also observed extended survival and better control of the challenge infection in the immunized mice.ConclusionsA robust anamnestic response following challenge including a Th-1 serum cytokine profile coupled with increased survival is indicative of protective immunity in the immunized mice. These observations suggest that β-tubulin of T. evansi is a viable antigenic target for development of a vaccine against this important livestock pathogen.