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High-level Expression (high-level + expression)
Selected AbstractsHigh-Level Expression of Proteins in Mammalian Cells Using Transcription Regulatory Sequences from the Chinese Hamster EF-1, GeneBIOTECHNOLOGY PROGRESS, Issue 3 2004Jennifer Running Deer High-level expression of a recombinant protein in Chinese hamster ovary (CHO) cells typically requires the laborious and time-consuming procedure of stepwise gene amplification. We hypothesized that use of transcription control regions from a highly expressed gene in CHO cells to drive expression of a gene of interest might reduce the requirement for gene amplification. To this end, we cloned a 19 kb DNA fragment containing the Chinese hamster elongation factor-1, (EF-1,) gene, as well as 12 kb of 5, flanking sequence and 4 kb of 3, flanking sequence. Expression vectors containing 5, and 3, flanking sequences from the Chinese hamster EF-1, (CHEF1) gene were constructed and, after insertion of six different reporter genes, transfected into CHO cells. For comparison, CHO cells were also transfected with the same six reporter genes inserted into commercial vectors utilizing either the immediate early promoter from cytomegalovirus (CMV) or the human EF-1, promoter. The striking result from these studies was that average expression levels from pooled, stable transfectants of CHEF1 vectors were 6- to 35-fold higher than expression levels from commercial vectors that utilize the CMV or the human EF-1, promoters. We also used a CHEF1 vector to express a secreted and a membrane-bound protein in stably transfected non-CHO cell lines. CHEF1-driven expression of secreted alkaline phosphatase (SEAP) in three of four cell lines tested (HEK 293, K562, L1.2, and HCT 116) was 13- to 280-fold greater than that from a commercial vector employing the CMV promoter. After transfection of four different cell lines of hematopoietic origin (K562, L1.2, JY, and Jurkat), the CHEF1 vector was found to express the chemokine receptor CCR4 at >10-fold higher levels than that driven from a commercial vector utilizing the CMV promoter. Results from these experiments suggest that the CHEF1 vectors will be useful for high-level protein expression not only in CHO cells, but also in a variety of other mammalian cell lines. [source] High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptorsPROTEIN SCIENCE, Issue 9 2010Zuzana Dostalova Abstract The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABAA receptors containing ,1 and ,3 subunits could be expressed with specific activities of 29,34 pmol/mg corresponding to 140,170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT3A) receptors were 49,63 pmol/mg and 245,315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3,3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one step after solubilization using ANTI-FLAG affinity chromatography with yields of 30,40%. Purified receptors were functional. Binding of the agonist [3H]muscimol to the purified GABAAR was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles. [source] High-Level Expression of Proteins in Mammalian Cells Using Transcription Regulatory Sequences from the Chinese Hamster EF-1, GeneBIOTECHNOLOGY PROGRESS, Issue 3 2004Jennifer Running Deer High-level expression of a recombinant protein in Chinese hamster ovary (CHO) cells typically requires the laborious and time-consuming procedure of stepwise gene amplification. We hypothesized that use of transcription control regions from a highly expressed gene in CHO cells to drive expression of a gene of interest might reduce the requirement for gene amplification. To this end, we cloned a 19 kb DNA fragment containing the Chinese hamster elongation factor-1, (EF-1,) gene, as well as 12 kb of 5, flanking sequence and 4 kb of 3, flanking sequence. Expression vectors containing 5, and 3, flanking sequences from the Chinese hamster EF-1, (CHEF1) gene were constructed and, after insertion of six different reporter genes, transfected into CHO cells. For comparison, CHO cells were also transfected with the same six reporter genes inserted into commercial vectors utilizing either the immediate early promoter from cytomegalovirus (CMV) or the human EF-1, promoter. The striking result from these studies was that average expression levels from pooled, stable transfectants of CHEF1 vectors were 6- to 35-fold higher than expression levels from commercial vectors that utilize the CMV or the human EF-1, promoters. We also used a CHEF1 vector to express a secreted and a membrane-bound protein in stably transfected non-CHO cell lines. CHEF1-driven expression of secreted alkaline phosphatase (SEAP) in three of four cell lines tested (HEK 293, K562, L1.2, and HCT 116) was 13- to 280-fold greater than that from a commercial vector employing the CMV promoter. After transfection of four different cell lines of hematopoietic origin (K562, L1.2, JY, and Jurkat), the CHEF1 vector was found to express the chemokine receptor CCR4 at >10-fold higher levels than that driven from a commercial vector utilizing the CMV promoter. Results from these experiments suggest that the CHEF1 vectors will be useful for high-level protein expression not only in CHO cells, but also in a variety of other mammalian cell lines. [source] Expression of a non-DNA-binding Ikaros isoform exclusively in B cells leads to autoimmunity but not leukemogenesisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2007Heather Wojcik Abstract Ikaros is a transcriptional regulator whose function is essential for B cell development. It is expressed in the hematopoietic stem cell (HSC) through the mature B cell stage. Using genetically engineered mice in which the endogenous Ikaros gene is disrupted, it has been shown that a lack of Ikaros leads to a block in B cell development and that its severe diminution results in a hyperresponsive B cell compartment. Ikaros expression within the HSC has led to speculation as to whether the role of Ikaros in B cell biology is largely accomplished prior to B cell specification. In addition, widespread expression of Ikaros in hematopoietic cells leads to the possibility that some or all of the observed defects are not B cell autonomous. In this report, we demonstrate that over-expression of a dominant interfering Ikaros isoform exclusively in B cells has profound effects on mature B cell function. We provide evidence that continued high-level expression of Ikaros is essential for homeostasis of peripheral lymphocytes and maintenance of B cell tolerance. We also show that deregulation of Ikaros activity does not rapidly result in B cell leukemogenesis as it does with 100% penetrance within the T cell lineage. [source] Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma,HEPATOLOGY, Issue 6 2006Boris Blechacz The oncolytic measles virus Edmonston strain (MV-Edm), a nonpathogenic virus targeting cells expressing abundant CD46, selectively destroys neoplastic tissue. Clinical development of MV-Edm would benefit from noninvasive monitoring strategies to determine the speed and extent of the spread of the virus in treated patients and the location of virus-infected cells. We evaluated recombinant MV-Edm expressing carcinoembryonic antigen (CEA) or the human sodium iodide symporter (hNIS) for oncolytic potential in hepatocellular carcinoma (HCC) and efficiency in tracking viruses in vivo by noninvasive monitoring. CD46 expression in human HCC and primary hepatocytes was assessed by flow cytometry and immunohistochemistry. Infectivity, syncytium formation, and cytotoxicity of recombinant MV-Edm in HCC cell lines were evaluated by fluorescence microscopy, crystal violet staining, and the MTS assay. Transgene expression in HCC cell lines after infection with recombinant MV-Edm in vitro and in vivo was assessed by CEA concentration, 125I-uptake, and 123I-imaging studies. Toxicology studies were performed in IfnarKO×CD46 transgenic mice. The CD46 receptor was highly expressed in HCC compared to nonmalignant hepatic tissue. Recombinant MV-Edm efficiently infected HCC cell lines, resulting in extensive syncytium formation followed by cell death. Transduction of HCC cell lines and subcutaneous HCC xenografts with recombinant MV-Edm resulted in high-level expression of transgenes in vitro and in vivo. MV-Edm was nontoxic in susceptible mice. Intratumoral and intravenous therapy with recombinant MV-Edm resulted in inhibition of tumor growth and prolongation of survival with complete tumor regression in up to one third of animals. In conclusion, engineered MV-Edm may be a potent and novel cancer gene therapy system for HCC. MV-Edm expressing CEA or hNIS elicited oncolytic effects in human HCC cell lines in vitro and in vivo, enabling the spread of the virus to be monitored in a noninvasive manner. (HEPATOLOGY 2006;44:1465,1477.) [source] Identification of a novel human tissue factor splice variant that is upregulated in tumor cells,INTERNATIONAL JOURNAL OF CANCER, Issue 7 2006Hitendra S. Chand Abstract Tissue factor (TF) is a transmembrane glycoprotein that serves as the prime initiator of blood coagulation and plays a critical role in thrombosis and hemostasis. In addition, a variety of tumor cells overexpress cell-surface TF, which appears to be important for tumor angiogenesis and metastasis. To elucidate the mechanism involved in the upregulation of TF in human tumor cells, a comprehensive analysis of TF mRNA from various normal and tumor cells was performed. The results of these studies indicate that, in addition to possessing a normal full-length TF transcript and minor levels of an alternatively spliced transcript known as alternatively-spliced tissue factor (asTF) (Bogdanov et al., Nat Med 2003;9:458,62), human tumor cells express additional full-length TF transcripts that are also generated by alternative splicing. Reverse transcriptase-polymerase chain reaction (RT-PCR) and 5,-rapid amplification of cDNA ends- (5,-RACE) based analyses of cytoplasmic RNA from normal and tumor cells revealed that there is alternative splicing of the first intron between exon I and exon II resulting in 2 additional TF transcripts. One of the transcripts has an extended exon I with inclusion of most of the first TF intron (955 bp), while the second transcript is formed by the insertion of a 495 bp sequence, referred to as exon IA, derived from an internal sequence of the first intron. The full length TF transcript with alternatively spliced novel exon IA, referred to as alternative exon 1A-tissue factor (TF-A), represented ,1% of the total TF transcripts in normal cells, but constituted 7,10% of the total TF transcript in tumor cells. Quantitative real-time RT-PCR analysis indicated that cultured human tumor cells contain 10,25-fold more copy numbers of TF-A in comparison to normal, untransformed cells. We propose that high-level expression of the novel TF-A transcript, preferentially in tumor cells, may have utility in the diagnosis and staging of a variety of solid tumors. © 2005 Wiley-Liss, Inc. [source] Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehiclesLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2008L.L. Fu Abstract Aims:, To achieve high-level expression and secretion of active VP28 directed by a processing-efficient signal peptide in Bacillus subtilis WB600 and exploit the possibility of obtaining an oral vaccine against white spot syndrome virus (WSSV) using vegetative cells or spores as delivery vehicles. Methods and Results:, The polymerase chain reaction (PCR)-amplified vp28 gene was inserted into a shuttle expression vector with a novel signal peptide sequence. After electro-transformation, time-courses for recombinant VP28 (rVP28) secretion level in B. subtilis WB600 were analysed. Crayfish were divided into three groups subsequently challenged by 7-h immersion at different time points after vaccination. Subgroups including 20 inter-moult crayfish with an average weight of 15 g in triplicate were vaccinated by feeding coated food pellets with vegetative cells or spores for 20 days. Vaccination trials showed that rVP28 by spore delivery induced a higher resistance than using vegetative cells. Challenged at 14 days postvaccination, the relative per cent survival (RPS) values of groups of rVP28-bv and rVP28-bs was 51·7% and 78·3%, respectively. Conclusions:, The recombinant B. subtilis strain with the ability of high-level secretion of rVP28 can evoke protection of crayfish against WSSV by oral delivery. Significance and Impact of the Study:, Oral vaccination by the B. subtilis vehicle containing VP28 opens a new way for designing practical vaccines to control WSSV. [source] Analysis of cis -regulatory elements in the 5, untranslated region of murine leukemia virus controlling protein expressionMICROBIOLOGY AND IMMUNOLOGY, Issue 3 2009Naoki Yamamoto ABSTRACT It has previously been reported by us that high-level expression of the Env protein of Fr-MLV clone A8 in brains is crucial for induction of spongiform neurodegeneration, and that the 0.3-kb fragment containing the R, U5, and the 5, leader sequence of A8 is responsible for neuropathogenicity. In the present study, the role of the 5, untranslated region in protein expression was investigated. Luciferase expression vectors containing the LTR (R-U3-U5) and 5, leader sequence of A8 and non-neuropathogenic 57 Fr-MLV, designated gl-A8 and gl-57, and their chimeric vectors, were constructed, and transfected into rat glial cells F10. Replacement of the region containing the 3, half of R, U5, and 5, leader sequence of gl-A8 with that of 57 showed a reduction in luciferase activities, and replacement of this region of gl-57 with that of A8 showed increased luciferase activity. These results show that the region containing the 3, half of R, U5, and 5, leader sequence of A8 more efficiently up-regulates protein expression than 57. In particular, the 3, half of 5, leader of A8 was most responsible for the up-regulation of protein expression. Of interest, after replacement of the fragments between A8 and 57, changes in the activities of vectors containing A8-U3 paralleled the amount of mRNA, but the activities of vectors containing 57-U3 did not. Furthermore, it is suggested that the region containing R, U5, and the 5, leader sequence influences transcriptional or post-transcriptional steps, depending on the upstream sequence containing enhancer elements and promoter. [source] The Ustilago maydis Cys2His2 -type zinc finger transcription factor Mzr1 regulates fungal gene expression during the biotrophic growth stageMOLECULAR MICROBIOLOGY, Issue 6 2008Yan Zheng§ Summary The smut fungus Ustilago maydis establishes a biotrophic relationship with its host plant maize to progress through sexual development. Here, we report the identification and characterization of the Cys2His2 -type zinc finger protein Mzr1 that functions as a transcriptional activator during host colonization. Expression of the U. maydis mig2 cluster genes is tightly linked to this phase. Upon conditional overexpression, Mzr1 confers induction of a subset of mig2 genes during vegetative growth and this requires the same promoter elements that confer inducible expression in planta. Furthermore, expression of the mig2-4 and mig2-5 genes during biotrophic growth is strongly reduced in cells deleted in mzr1. DNA-array analysis led to the identification of additional Mzr1-induced genes. Some of these genes show a mig2 -like plant-specific expression pattern and Mzr1 is responsible for their high-level expression during pathogenesis. Mzr1 function requires the b -dependently regulated Cys2His2 -type cell cycle regulator Biz1, indicating that two stage-specific regulators mediate gene expression during host colonization. In spite of a role as transcriptional activator during biotrophic growth, mzr1 is not essential for pathogenesis; however, conditional overexpression interfered with proliferation during vegetative growth and mating ability, caused a cell separation defect, and triggered filamentous growth. We discuss the implications of these findings. [source] Leaving on the lights: host-specific derepression of Mycobacterium tuberculosis gene expression by anti-sigma factor gene mutationsMOLECULAR MICROBIOLOGY, Issue 5 2006Robert N. Husson Summary Regulation of transcription by alternative sigma factors is a strategy widely used by bacteria to adapt to changes in environmental conditions. For several pathogenic bacteria, alternative sigma factor-regulated gene expression is critical for virulence. The activity of many alternative sigma factors is in turn controlled by regulatory proteins that transduce and integrate environmental signals. In this issue of Molecular Microbiology, Said-Salim et al. demonstrate high-level expression of genes encoding major protein antigens in the bovine subspecies of Mycobacterium tuberculosis, in contrast to low-level expression in the human subspecies. Having previously suggested that SigK regulates the expression of these genes, the authors found that the high-expressers have point mutations in Rv0444c, a gene adjacent to sigK, and provided evidence that this gene encodes an anti-sigma factor whose function is abrogated by these mutations. These findings not only demonstrate an adaptive mechanism of potential importance in tuberculosis immunity and pathogenesis, but also raise interesting questions regarding the origin of these mutations and their effects on anti-sigma factor function. [source] Identification and gene expression profiling of the Pum1 and Pum2 members of the Pumilio family in the chickenMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2008Jee Young Lee Abstract Members of the Pumilio (Pum) family of RNA-binding proteins act as translational repressors and are required for germ cell development and asymmetric division. We identified the chicken Pum1 and Pum2 genes and analyzed their expression patterns in various tissues. Comparative sequence analysis of the Pum1 and Pum2 proteins from the drosophila, chicken, mouse, and human revealed a high degree of evolutionary conservation in terms of the levels of homology of the peptide sequences and the structure of Pumilio homology domain (PUM-HD), C-terminal RNA-binding domain, with similar spacing between the adjacent Pum eight tandem repeats. In addition, phylogenetic patterns of pumilio family showed that Pum 1 and 2 of chicken are more closely related to those of mouse and human than other species and Pum1 is more conserved than Pum2. Using real-time RT-PCR, the expression levels of the Pum1 and Pum2 genes were found to be highest in hatched female gonads, and high-level expression of Pum2 was detected in 12-day and hatched gonads among the various chicken embryonic tissues tested. In adult tissues, the expression levels of Pum1 and Pum2 were expressed at higher levels in the testis and muscle than in any other tissue. The characteristics of the tissue-specific expression of Pum genes suggest that Pum1 and Pum2 have effects crucially in particular stage during development of chicken gonads depending on sexual maturation. Mol. Reprod. Dev. 75: 184,190, 2008. © 2007 Wiley-Liss, Inc. [source] High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptorsPROTEIN SCIENCE, Issue 9 2010Zuzana Dostalova Abstract The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABAA receptors containing ,1 and ,3 subunits could be expressed with specific activities of 29,34 pmol/mg corresponding to 140,170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT3A) receptors were 49,63 pmol/mg and 245,315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3,3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one step after solubilization using ANTI-FLAG affinity chromatography with yields of 30,40%. Purified receptors were functional. Binding of the agonist [3H]muscimol to the purified GABAAR was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles. [source] FGFR3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer,THE JOURNAL OF PATHOLOGY, Issue 1 2007DC Tomlinson Abstract FGFR3 is frequently activated by mutation in urothelial carcinoma (UC) and represents a potential target for therapy. In multiple myeloma, both over-expression and mutation of FGFR3 contribute to tumour development. To define the population of UC patients who may benefit from FGFR-targeted therapy, we assessed both mutation and receptor over-expression in primary UCs from a population of new patients. Manual or laser capture microdissection was used to isolate pure tumour cell populations. Where present, non-invasive and invasive components in the same section were microdissected. A screen of the region of the highest tumour stage in each sample yielded a mutation frequency of 42%. Mutations comprised 61 single and five double mutations, all in hotspot codons previously identified in UC. There was a significant association of mutation with low tumour grade and stage. Subsequently, non-invasive areas from the 43 tumours with both non-invasive and invasive components were analysed separately; 18 of these had mutation in at least one region, including nine with mutation in all regions examined, eight with mutation in only the non-invasive component and one with different mutations in different regions. Of the eight with mutation in only the non-invasive component, six were predicted to represent a single tumour and two showed morphological dissimilarity of fragments within the block, indicating the possible presence of distinct tumour clones. Immunohistochemistry showed over-expression of FGFR3 protein in many tumours compared to normal bladder and ureteric controls. Increased expression was associated with mutation (85% of mutant tumours showed high-level expression). Overall, 42% of tumours with no detectable mutation showed over-expression, including many muscle-invasive tumours. This may represent a non-mutant subset of tumours in which FGFR3 signalling contributes to the transformed phenotype and which may benefit from FGFR-targeted therapies. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] A novel synthetic mammalian promoter derived from an internal ribosome entry siteBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2006Shizuka Hartenbach Abstract Introduction of specific mutations into a synthetic internal ribosome entry site (IRESGTX) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic PGTX promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high-level expression of a variety of transgenes including the human vascular endothelial growth factor 121 (VEGF121), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus -derived secreted ,-amylase (SAMY) in Chinese hamster ovary cells (CHO-K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleotide (resulting in PGTX2) doubled the SEAP expression levels in CHO-K1. PGTX2 reached near 70% of PSV40 -driven expression levels and outperformed constitutive phosphoglycerate kinase (PPGK) and human ubiquitin C (PhUBC) promoters in CHO-K1. Also, PGTX2 was successfully engineered for macrolide-inducible transgene expression. Owing to its size of only 182 bp, PGTX2 is one of the smallest eukaryotic promoters. Although PGTX2 was found to be a potent promoter, it retained its IRESGTX -specific translation-initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells. © 2006 Wiley Periodicals, Inc. [source] Chemokine receptors in cancer metastasis and cancer cell-derived chemokines in host immune responseCANCER SCIENCE, Issue 11 2007Keiichi Koizumi The chemotactic cytokines called chemokines are a superfamily of small secreted cytokines that were initially characterized through their ability to prompt the migration of leukocytes. Attention has been focused on the chemokine receptors expressed on cancer cells because cancer cell migration and metastasis show similarities to leukocyte trafficking. CXC chemokine receptor 4 (CXCR4) was first investigated as a chemokine receptor that is associated with lung metastasis of breast cancers. Recently, CXCR4 was reported to be a key molecule in the formation of peritoneal carcinomatosis in gastric cancer. In the present review, we highlight current knowledge about the role of CXCR4 in cancer metastases. In contrast to chemokine receptors expressed on cancer cells, little is known about the roles of cancer cell-derived chemokines. Cancer tissue consists of both cancer cells and various stromal cells, and leukocytes that infiltrate into cancer are of particular importance in cancer progression. Although colorectal cancer invasion is regulated by the chemokine CCL9-induced infiltration of immature myeloid cells into cancer, high-level expression of cancer cell-derived chemokine CXCL16 increases infiltrating CD8+ and CD4+ T cells into cancer tissues, and correlates with a good prognosis. We discuss the conflicting biological effects of cancer cell-derived chemokines on cancer progression, using CCL9 and CXCL16 as examples. (Cancer Sci 2007; 98: 1652,1658) [source] | |||||||||||||