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Efficient Expression (efficient + expression)
Selected AbstractsEfficient expression and purification of human aglycosylated Fc, receptors in Escherichia coli,BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Sang Taek Jung Abstract Effector Fc gamma receptors (Fc,Rs) are expressed on the surface of a variety of cells of hematopoietic lineage and serve as a bridge between adaptive and innate immune responses. The interaction between immune complexes, formed by IgG class antibodies that are crosslinked with antigen, and Fc,Rs triggers signaling cascades that result in numerous cellular responses including the activation or donwregulation of cytotoxic responses, cytokine release, and antibody synthesis. Here, the extracellular domains of the human type I transmembrane Fc,Rs were expressed in Escherichia coli and their interactions to subclass IgGs (IgG1, IgG2, IgG3, and IgG4) antibodies were analyzed. Expression using fully synthetic E. coli codon optimized Fc,R genes and optimization of sequences for N-terminal translation initiation region through mRNA secondary structure prediction enabled us to achieve high yield of purified, bacterially expressed receptors, including Fc,RI and Fc,RIIIa which have not been successfully expressed in bacteria until now. The aglycosylated Fc,Rs showed similar IgG subclass binding selectivity compared to the respective glycosylated Fc,Rs expressed in mammalian cells. Biotechnol. Bioeng. 2010;107: 21,30. © 2010 Wiley Periodicals, Inc. [source] Indications for cell stress in response to adenoviral and baculoviral gene transfer observed by proteome profiling of human cancer cellsELECTROPHORESIS, Issue 11 2010Christopher Gerner Abstract Gene transfer to cultured cells is an important tool for functional studies in many areas of biomedical research and vector systems derived from adenoviruses and baculoviruses are frequently used for this purpose. In order to characterize how viral gene transfer vectors affect the functional state of transduced cells, we applied 2-D PAGE allowing quantitative determination of protein amounts and synthesis rates of metabolically labeled cells and shotgun proteomics. Using HepG2 human hepatoma cells we show that both vector types can achieve efficient expression of green fluorescent protein, which accounted for about 0.1% of total cellular protein synthesis 72,h after transduction. No evidence in contrast was found for expression of proteins from the viral backbones. With respect to the host cell response, both vectors induced a general increase in protein synthesis of about 50%, which was independent of green fluorescent protein expression. 2-D PAGE autoradiographs identified a 3.6-fold increase of ,-actin synthesis in adenovirus transduced cells. In addition shotgun proteomics of cytoplasmic and nuclear extract fractions identified a slight induction of several proteins related to inflammatory activation, cell survival and chromatin function by both virus types. These data demonstrate that commonly used gene transfer vectors induce a response reminiscent of stress activation in host cells, which needs to be taken into account when performing functional assays with transduced cells. [source] Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surfaceJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 4 2010Irina V. Balyasnikova Abstract Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright © 2009 John Wiley & Sons, Ltd. [source] Dendritic cells lentivirally engineered to overexpress interleukin-10 inhibit contact hypersensitivity responses, despite their partial activation induced by transduction-associated physical stressTHE JOURNAL OF GENE MEDICINE, Issue 3 2010Verena Besche Abstract Background Dendritic cells (DCs) constitute an attractive target for immunotherapeutic approaches. Because DCs are largely refractory to transfection with plasmid DNA, several viral transduction protocols were established. The potential side-effects of lentiviral transduction on the phenotype and activation state of DCs left unstimulated after transduction have not been assessed. There is a need to analyse these parameters as a result of the requirement of using DCs with a low activation state for therapeutic strategies intended to induce tolerance. Methods Lentivirally-transduced bone marrow (BM)-derived DCs (LV-DCs) in comparison with mock-transduced (Mock-DCs) and untreated DCs were analysed with regard to the induction of maturation processes on the RNA, protein and functional level. BM-DCs engineered to overexpress interleukin (IL)-10 were analysed for therapeutic potential in a mouse model of allergic contact dermatitis. Results Compared with untreated DCs, Mock-DCs and LV-DCs displayed an altered gene expression signature. Mock-DCs induced a stronger T cell proliferative response than untreated DCs. LV-DCs did not further augment the T cell proliferative response, but induced a slightly different T cell cytokine pattern compared to Mock-DCs. Accordingly, the gene promoter of the DC maturation marker fascin mediated efficient expression of the model transgene IL-10 in unstimulated-transduced BM-DCs. Nevertheless, IL-10 overexpressing BM-DCs exerted tolerogenic activity and efficiently inhibited the contact hypersensitivity response in previously hapten-sensitized mice. Conclusions Lentiviral transduction of BM-DCs results in their partial activation. Nevertheless, the transduction of these DCs with a vector encoding the immunomodulatory cytokine IL-10 rendered them tolerogenic. Thus, lentivirally-transduced DCs expressing immunomodulatory molecules represent a promising tool for induction of tolerance. Copyright © 2010 John Wiley & Sons, Ltd. [source] A retrovirus-based system to stably silence GDF-8 expression and enhance myogenic differentiation in human rhabdomyosarcoma cellsTHE JOURNAL OF GENE MEDICINE, Issue 8 2008Zhuo Yang Abstract Background Myostatin, also called GDF-8, a secreted growth and differentiating factor that belongs to the transforming growth factor-, superfamily, is a known negative regulator of myogenesis in vivo. Overexpression of GDF-8 contributes to the lack of differentiation in human rhabdomyosarcoma (RMS) cells. We investigated whether a retrovirus-based RNA interference (RNAi) system against GDF-8 expression in human RMS cells would enhance myogenic differentiation. Methods A retrovirus-based RNAi system was developed that utilized the U6-RNA polymerase III promoter to drive efficient expression and deliver the GDF8-specific short hairpin RNAs (shRNAs) in human RMS cell A204. In this system, the retrovirus vector was integrated into the host cell genome and allowed stable expression of shRNAs. GDF-8 expression was determined by real-time polymerase chain reaction and western blotting analysis. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cell proliferation. Myogenic differentiation markers were monitored by western blotting analysis. Cell cycle and apoptosis was determined by propidium iodide staining and analysed in a flow cytometer. Results In the siGDF8 A204 cell pools, the levels of both GDF-8 mRNA and protein were dramatically reduced by this RNAi system. In differentiation conditions, inhibition of myostatin synthesis led to enhanced cell cycle withdrawal, consequently stimulated myogenic differentiation and increased the rate of tumor cell apoptosis. Conclusions The results demonstrate that deactivation of myostatin by using retrovirus-based RNAi thus may be useful for therapy in rhabdomyosarcomas. Copyright © 2008 John Wiley & Sons, Ltd. [source] Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoproteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Jens Hausmann Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an ,100,kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0,Å resolution and allowed the collection of complete diffraction data to about 2.6,Å resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed. [source] Computationally efficient expressions for the collision efficiency between electrically charged aerosol particles and cloud dropletsTHE QUARTERLY JOURNAL OF THE ROYAL METEOROLOGICAL SOCIETY, Issue 618 2006S. N. Tripathi Abstract A multiple factor parametrization is described to permit the efficient calculation of collision efficiency (E) between electrically charged aerosol particles and neutral cloud droplets in numerical models of cloud and climate. The four-parameter representation summarizes the results obtained from a detailed microphysical model ofE, which accounts for the different forces acting on the aerosol in the path of falling cloud droplets. The parametrization's range of validity is for aerosol particle radii of 0.4 to 10 ,m, aerosol particle densities of 1 to 2.0 g cm,3, aerosol particle charges from neutral to 100 elementary charges and drop radii from 18.55 to 142 , m. The parametrization yields values ofE well within an order of magnitude of the detailed model's values, from a dataset of 3978E values. Of these values 95% have modelled to parametrized ratios between 0.5 and 1.5 for aerosol particle sizes ranging between 0.4 and 2.0 , m, and about 96% in the second size range. This parametrization speeds up the calculation ofE by a factor of ,103 compared with the original microphysical model, permitting the inclusion of electric charge effects in numerical cloud and climate models. Copyright © 2006 Royal Meteorological Society [source] |