EIF4A2 as another novel fusion partner of PLAG1 in lipoblastoma

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

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A Novel NFIX-STAT6 Gene Fusion in Solitary Fibrous Tumor: A Case Report

International Journal of Molecular Sciences ◽

10.3390/ijms22147514 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7514

Author(s):

David S. Moura ◽

Juan Díaz-Martín ◽

Silvia Bagué ◽

Ruth Orellana-Fernandez ◽

Ana Sebio ◽

...

Keyword(s):

Solitary Fibrous Tumor ◽

Gene Fusion ◽

Wide Spectrum ◽

Bioinformatic Analysis ◽

Fusion Partner ◽

Rna Seq ◽

Genomic Location ◽

Nuclear Factor I ◽

New Gene ◽

Fibrous Tumor

Solitary fibrous tumor is a rare subtype of soft-tissue sarcoma with a wide spectrum of histopathological features and clinical behaviors, ranging from mildly to highly aggressive tumors. The defining genetic driver alteration is the gene fusion NAB2–STAT6, resulting from a paracentric inversion within chromosome 12q, and involving several different exons in each gene. STAT6 (signal transducer and activator of transcription 6) nuclear immunostaining and/or the identification of NAB2–STAT6 gene fusion is required for the diagnostic confirmation of solitary fibrous tumor. In the present study, a new gene fusion consisting of Nuclear Factor I X (NFIX), mapping to 19p13.2 and STAT6, mapping to 12q13.3 was identified by targeted RNA-Seq in a 74-year-old female patient diagnosed with a deep-seated solitary fibrous tumor in the pelvis. Histopathologically, the neoplasm did not display nuclear pleomorphism or tumor necrosis and had a low proliferative index. A total of 378 unique reads spanning the NFIXexon8–STAT6exon2 breakpoint with 55 different start sites were detected in the bioinformatic analysis, which represented 59.5% of the reads intersecting the genomic location on either side of the breakpoint. Targeted RNA-Seq results were validated by RT-PCR/ Sanger sequencing. The identification of a new gene fusion partner for STAT6 in solitary fibrous tumor opens intriguing new hypotheses to refine the role of STAT6 in the sarcomatogenesis of this entity.

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Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2018-0441-oa ◽

2019 ◽

Vol 144 (1) ◽

pp. 90-98 ◽

Cited By ~ 5

Author(s):

Robyn T. Sussman ◽

Amanda R. Oran ◽

Carmela Paolillo ◽

David Lieberman ◽

Jennifer J. D. Morrissette ◽

...

Keyword(s):

Next Generation Sequencing ◽

Solid Tumors ◽

Gold Standard ◽

University Hospital ◽

Fusion Partner ◽

Next Generation ◽

Fixed Tissue ◽

Fusion Transcripts ◽

Genetic Abnormalities ◽

Generation Sequencing

Context.— Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK. Objective.— To validate an anchored multiplex polymerase chain reaction–based panel for the detection of fusion transcripts in a university hospital–based clinical molecular diagnostics laboratory. Design.— We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffin-embedded specimens with as little as 10% tumor nuclei. Results.— Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant III (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific. Conclusions.— This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors.

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The serum albumin-binding region of streptococcal protein G: a bacterial fusion partner with carrier-related properties

Journal of Immunological Methods ◽

10.1016/s0022-1759(96)00217-7 ◽

1997 ◽

Vol 201 (1) ◽

pp. 115-123 ◽

Cited By ~ 42

Author(s):

Anders Sjölander ◽

Per-Åke Nygren ◽

Stefan Ståhl ◽

Klavs Berzins ◽

Mathias Uhlén ◽

...

Keyword(s):

Serum Albumin ◽

Protein G ◽

Albumin Binding ◽

Fusion Partner ◽

Binding Region ◽

Streptococcal Protein G

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Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR

Applied and Environmental Microbiology ◽

10.1128/aem.70.8.4582-4587.2004 ◽

2004 ◽

Vol 70 (8) ◽

pp. 4582-4587 ◽

Cited By ~ 112

Author(s):

Jan Kostal ◽

Rosanna Yang ◽

Cindy H. Wu ◽

Ashok Mulchandani ◽

Wilfred Chen

Keyword(s):

Cell Growth ◽

Environmental Protection Agency ◽

Arsenic Removal ◽

Fusion Partner ◽

Resting Cells ◽

Bacterial Cells ◽

High Affinity ◽

Affinity Ligand ◽

Arsenic Accumulation ◽

Severe Reduction

ABSTRACT The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd2+ or Zn2+ accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.

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Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy

Molecules ◽

10.3390/molecules26133936 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3936

Author(s):

Svetlana I. Bakholdina ◽

Anna M. Stenkova ◽

Evgenia P. Bystritskaya ◽

Evgeniy V. Sidorin ◽

Natalya Yu. Kim ◽

...

Keyword(s):

Inclusion Bodies ◽

Yersinia Pseudotuberculosis ◽

Fluorescent Protein ◽

Chimeric Protein ◽

Maximum Level ◽

Molecular Organization ◽

Eukaryotic Cells ◽

Fusion Partner ◽

Phospholipase A1

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

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Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽

10.1210/endo.138.2.4920 ◽

1997 ◽

Vol 138 (2) ◽

pp. 588-593 ◽

Cited By ~ 8

Author(s):

Y. Bobovnikova ◽

P. N. Graves ◽

H. Vlase ◽

T. F. Davies

Keyword(s):

Amino Acids ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Thioredoxin Reductase ◽

Biologically Active ◽

Receptor Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Fusion Partner ◽

E Coli ◽

Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

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Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Microbial Cell Factories ◽

10.1186/s12934-020-01502-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Young Su Kim ◽

Hye-Jeong Lee ◽

Man-ho Han ◽

Nam-kyung Yoon ◽

Yeu-chun Kim ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Factors ◽

Growth Factor ◽

Fusion Proteins ◽

Human Growth ◽

Soluble Form ◽

Fusion Partner ◽

Small Protein ◽

E Coli ◽

Tev Protease

Abstract Background Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. Results We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. Conclusions We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

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Intramuscular Tenosynovial Giant Cell Tumor Harboring a Novel CSF1-CD96 Fusion Transcript

International Journal of Surgical Pathology ◽

10.1177/10668969211049833 ◽

2021 ◽

pp. 106689692110498

Author(s):

Haider Mejbel ◽

Gene P. Siegal ◽

Shi Wei

Keyword(s):

Giant Cell Tumor ◽

Giant Cell ◽

Gene Rearrangement ◽

Fusion Transcript ◽

Cell Tumor ◽

Molecular Profile ◽

Small Subset ◽

Fusion Partner ◽

Giant Cell Tumors ◽

Tenosynovial Giant Cell Tumor

Tenosynovial giant cell tumors typically arise in the synovium of joints, bursae, or tendon sheaths. They may occur in an intra- or extra-articular location and can be divided into localized and diffuse types. The neoplastic nature of the lesion has been supported by a recurrent CSF1 gene rearrangement in a small subset of lesional cells, of which the most common fusion partner is COL6A3. Herein, we report a case of intramuscular localized tenosynovial giant cell tumor harboring a novel CSF1-CD96 fusion transcript, thus expanding the molecular profile of this tumor.

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