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Baculovirus Expression System (baculoviru + expression_system)
Selected AbstractsCharacterization of a novel silkworm (Bombyx mori) phenol UDP-glucosyltransferaseFEBS JOURNAL, Issue 3 2002Teresa Luque Sugar conjugation is a major pathway for the inactivation and excretion of both endogenous and exogenous compounds. We report here the molecular cloning and functional characterization of a phenol UDP-glucosyltransferase (UGT) from the silkworm, Bombyx mori, which was named BmUGT1. The complete cDNA clone is 1.6 kb, and the gene is expressed in several tissues of fifth-instar larvae, including fat body, midgut, integument, testis, silk gland and haemocytes. The predicted protein comprises 520 amino acids and has ,,30% overall amino-acid identity with other members of the UGT family. The most conserved region of the protein is the C-terminal half, which has been implicated in binding the UDP-sugar. BmUGT1 was expressed in insect cells using the baculovirus expression system, and a range of compounds belonging to diverse chemical groups were assessed as potential substrates for the enzyme. The expressed enzyme had a wide substrate specificity, showing activity with flavonoids, coumarins, terpenoids and simple phenols. These results support a role for the enzyme in detoxication processes, such as minimizing the harmful effects of ingested plant allelochemicals. This work represents the first instance where an insect ugt gene has been associated with a specific enzyme activity. [source] The interferon alpha induced protein ISG12 is localized to the nuclear membraneFEBS JOURNAL, Issue 22 2001Pia M. Martensen Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon , inducible genes of unknown function. We have determined the 5, end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon , inducibility of the gene. Subsequently, we have expressed human ISG12, a 12-kDa hydrophobic protein in the baculovirus expression system and with a C-terminal FLAG-tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon , by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope. [source] Enhancement of protective humoral immune responses against Herpes simplex virus-2 in DNA-immunized guinea-pigs using protein boostingFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2008Fatemeh Fotouhi Abstract Genital Herpes is a common sexually transmitted disease that is caused mostly by Herpes simplex virus type 2 (HSV-2). Its prevalence has increased in developing countries in spite of the availability of valuable antiviral drug therapy. Considering the importance of HSV-2 infections, effective vaccines remain the most likely hope for controlling the spread of HSV diseases. In the present study, the complete HSV-2 glycoprotein D gene was isolated and cloned into different plasmid vectors to construct a DNA vaccine and prepare recombinant subunit vaccines using a baculovirus expression system. The vaccines were tested alone or in combination to evaluate their ability to induce protective immunity in guinea-pigs against genital HSV infections. Immunization elicited humoral responses as measured by neutralization tests and enzyme-linked immunosorbent assay, and immunized animals had less severe genital skin disease as well as reduced replication of the challenging virus in the genital tract during experimental infection. Our results further demonstrate that DNA priming-protein boosting induced a neutralizing antibody titer higher than that obtained with DNA,DNA vaccination. The massive increase of antibody titer following DNA priming-protein boosting might be attributed to a recall of B cell memory. [source] The reconstituted human Chl12-RFC complex functions as a second PCNA loaderGENES TO CELLS, Issue 4 2004Yasushi Shiomi The sister chromatid cohesion factor Chl12 shares amino acid sequence similarity with RFC1, the largest subunit of replication factor C (RFC), and forms a clamp loader complex in association with the RFC small subunits RFCs2-5. It has been shown that the human Chl12-RFC complex, reconstituted with a baculovirus expression system, specifically interacts with human proliferating cell nuclear antigen (PCNA). The purified Chl12-RFC complex is structurally indistinguishable from RFC, as shown by electron microscopy, and it exhibits DNA-stimulated ATPase activity that is further enhanced by PCNA, and by DNA binding activity on specific primer/template DNA structures. Furthermore, the complex loads PCNA onto a circular DNA substrate, and stimulates DNA polymerase , DNA synthesis on a primed M13 single-stranded template in the presence of purified replication proteins. However, it cannot substitute for RFC in promoting simian virus 40 DNA replication in vitro with crude fractions. These results demonstrate that the human Chl12-RFC complex is a second PCNA loader and that its roles in replication are clearly distinguishable from those of RFC. [source] Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylatedINSECT MOLECULAR BIOLOGY, Issue 3 2010K. Moriya Abstract The lipid modifications which occur on Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 were studied by metabolic labelling in an insect cell-free protein synthesis system and in a baculovirus expression system, using specific inhibitors of protein prenylation and protein palmitoylation. In addition, the subcellular localization of BmRas proteins was examined using EGFP fusion proteins of constitutively active forms of BmRas proteins transiently expressed in Sf9 cells. As a result, it was revealed that the three B. mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylated for localization to the plasma membrane of insect cells. Thus, the mechanism of membrane binding of insect Ras proteins is quite different from that reported for mammalian Ras proteins. [source] Molecular cloning of the cDNAs encoding the feline B-lymphocyte activation antigen B7,1 (CD80) and B7,2 (CD86) homologues which interact with human CTLA4-IgINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 5-6 2000Y. Nishimura We cloned the cDNAs encoding the feline homologues of B-lymphocyte activation antigens B7,1 (CD80) and B7,2 (CD86). We expressed recombinant feline CD80 and CD86 molecules by the baculovirus expression system, and demonstrated their binding ability to human CTLA4-murine immunoglobulin fusion protein. [source] Two homologous parasitism-specific proteins encoded in Cotesia plutellae bracovirus and their expression profiles in parasitized Plutella xylostellaARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2008Sunyoung Lee Abstract A wasp, Cotesia plutellae, parasitizes the diamondback moth, Plutella xylostella, and interrupts host physiology for wasp survival and development. Identification of parasitism-specific factors would be helpful to understand the host,parasitoid interaction. This study focused on identification of a 15-kDa protein found only in plasma of the parasitized P. xylostella. Degenerate primers were designed after N-terminal amino acid sequencing of the parasitism-specific protein and used to clone the corresponding gene from the parasitized P. xylostella by a nested reverse transcriptase-polymerase chain reaction (RT-PCR). Two homologous genes were cloned and identified as "CpBV15," and "CpBV15,," respectively, due to the identical size (158 amino acid residues) of the predicted open reading frames, in which they shared amino acid sequences in both terminal regions, but varied in internal sequences. Southern hybridization analysis indicated that both genes were located on C. plutellae bracovirus genome. Real-time quantitative RT-PCR revealed that both genes were mostly expressed at the late parasitization period, which was further confirmed by an immunoblotting assay using CpBV15 antibody. A recombinant CpBV15 protein was produced from Sf9 cells via a baculovirus expression system. The purified CpBV15 protein could enter hemocytes of P. xylostella and were localized in the cytosol. Along with the sequence similarities of CpBV15s with eukaryotic initiation factors, their putative biological role has been discussed in terms of the host translation inhibitory factor. Arch. Insect Biochem. Physiol. 67:157,171, 2008. © 2008 Wiley-Liss, Inc. [source] Functional analysis of four Gloverin -like genes in the silkworm, Bombyx moriARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2008Shinpei Kawaoka Abstract To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli -challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin -like genes, BmGlov1,4, in the Bombyx genome. Northern blot and RT,PCR analysis showed that BmGlov1,4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-,B binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1,4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1,4 significantly inhibited the growth of E. coli. Arch. Insect Biochem. Physiol. 2007. © 2007 Wiley-Liss, Inc. [source] Site-specific unglycosylation to improve crystallization of the metabotropic glutamate receptor 3 extracellular domainACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Takanori Muto Metabotropic glutamate receptors (mGluRs) are involved in the regulation of many physiological and pathological processes in the central nervous system. The extracellular domain (ECD) of mGluR subtype 3 (mGluR3) was produced using the baculovirus expression system and purified from the culture medium. However, the recombinant protein showed heterogeneity in molecular weight on SDS,PAGE analysis. It was found that the unglycosylation of Asn414 significantly reduced the heterogeneity. Consequently, three site-specifically unglycosylated mutant proteins of mGluR3 ECD, replacing Asn414 only or replacing Asn414 in combination with other glycosylation sites, were successfully crystallized in the presence of l -glutamate. Among them, crystals of the N414/439Q mutant diffracted X-rays to 2.35,Å resolution using synchrotron radiation. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 84.0, b = 97.5, c = 108.1,Å, , = 93.0°. Assuming the presence of two protomers per crystallographic asymmetric unit, the Matthews coefficient VM was calculated to be 3.5,Å3,Da,1 and the solvent content was 65%. [source] Enhanced kinetic extraction of parvovirus B19 structural proteinsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2002Colleen Sico Abstract Recombinant structural proteins (VP1 and VP2) of the human parvovirus B19 have been expressed simultaneously using the baculovirus expression system to form virus-like particles (VLPs) that have potential use as vaccines. In this study, we report optimization of extraction conditions to recover these VLPs from cell paste. Under hypotonic conditions with neutral pH these VLPs were poorly extracted (up to 3% extraction). Addition of reducing agents, detergents, salts, and sonication did not improve the extractability. While screening for conditions to improve the extractability of the VLPs, we discovered that a combination of higher pH and elevated processing temperature significantly increased the extraction. Whereas increasing pH alone increased extractability from 3% to 6% (pH increased from 8.0 to 9.5), the effect of elevated temperature was much more substantial. At 50°C, we observed the extraction to be more than fivefold higher than that at room temperature (up to 25% extracted at pH 9.0). The kinetics of extraction at elevated temperatures showed a rapid initial rate of extraction (on the order of minutes) followed by a plateau. In addition, we compared the extraction of VP1 expressed alone. VP1 expressed alone is incapable of forming VLPs. We observed that non-VLP VP1 was easily extractable (up to 60% extracted) under conditions in which the VP1 + VP2 VLPs were not extractable. From these studies we conclude that parvovirus B19 structural proteins expressed to form VLPs have a hindered extractability as compared with non-VLP protein. This hindrance to extraction can be significantly reduced by processing at elevated temperatures and an increased pH, possibly due to the enhanced rates of solubilization and diffusion. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng80: 250,256, 2002. [source] Mouse × pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodiesBIOTECHNOLOGY PROGRESS, Issue 2 2009Ana M. Jar Abstract The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse × pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Expression, purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of metabotropic glutamate receptor 7ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2007Takanori Muto Glutamate is the major excitatory neurotransmitter and its metabotropic glutamate receptor (mGluR) plays an important role in the central nervous system. The ligand-binding domain (LBD) of mGluR subtype 7 (mGluR7) was produced using the baculovirus expression system and purified from the culture medium. The purified protein was characterized by gel-filtration chromatography, SDS,PAGE and a ligand-binding assay. Crystals of mGluR7 LBD were grown at 293,K by the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 3.30,Å resolution using synchrotron radiation and belong to the trigonal space group P3121, with unit-cell parameters a = b = 92.4, c = 114.3,Å. Assuming the presence of one protomer per crystallographic asymmetric unit, the Matthews coefficient VM was calculated to be 2.5,Å3,Da,1 and the solvent content was 51%. [source] Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systemsEQUINE VETERINARY JOURNAL, Issue 7 2001E. L. J. McMONAGLE Summary Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-, production in cells derived from equine lymph nodes. Preincubation of IFN-, inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-, induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-, production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species. [source] Identification of genes encoding N -glycan processing ,- N -acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systemsBIOTECHNOLOGY PROGRESS, Issue 1 2010Christoph Geisler Abstract Glycoproteins produced by non-engineered insects or insect cell lines characteristically bear truncated, paucimannose N -glycans in place of the complex N -glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N -glycan processing ,- N -acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N -glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing ,- N -acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Bioinformatic analyses of putative processing ,- N -acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing ,- N -acetylglucosaminidases than degradative or chitinolytic ,- N -acetylglucosaminidases. In addition, over-expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing ,- N -acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus-insect cell expression systems for the production of glycosylated proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | ||||||||||||||||