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dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge.in order to test the feasibility of baculovirus (autographa californica nuclear polyhedrosis virus, acnpv) expression vectors for making immunogens against dengue-1 (den-1) virus, a portion of the envelope (e) glycoprotein gene of den-1 virus was cloned and expressed. the recombinant baculovirus contains 107 nucleotides from the 3' terminus of the den-1 matrix (m) gene, which encodes a hydrophobic signal peptide and extends through the first 1, 245 nucleotides of e, terminating 243 nucleotides b ...19911877710
cysteine-86 is not needed for the enzymic activity of glutathione s-transferase 3-3.recombinant glutathione s-transferase 3-3 expressed in spodoptera frugiperda (sf9) cells with the use of a baculovirus expression system was modified with 1 mm-iodoacetamide. amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. the s-carbaminomethylated protein retains the gsh-conjugating activity. glutathione s-transferase 3-3 modified with iodo[14c]acetamide was digested with achromobacter proteinase i and the resulting peptides were separated by h ...19911883338
biological function of recombinant il-6 expressed in a baculovirus system.the cdna of human interleukin-6 (il-6) was cloned into baculovirus dna. insect cells (spodoptera frugiperda cells) infected with the recombinant baculovirus secreted a large amount of 22k protein into the culture medium. this culture fluid contained high biological activity of growth stimulation of a mouse myeloid cell line (mh-60). the il-6 was purified by a one-step procedure employing immunoaffinity chromatography of monoclonal antibody to il-6. the specific activities (bsf-2 reference units/ ...19911883915
characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal antibody specific for human fibrin fragment d-dimer.a recombinant single-chain molecule, scfv-k12g0, containing the variable domains of the monoclonal antibody ma-15c5, specific for fragment d-dimer of human cross-linked fibrin, was constructed and expressed in spodoptera frugiperda, sf9, insect cells. the arg108 carboxyl-terminal amino acid of the variable domain of the light-chain of the antibody was connected through a synthetic ala-gly-gln-gly-ser-ser-val peptide linker with the gln1 amino-terminal amino acid of the variable domain of its hea ...19911885569
synthesis of the virus-specified tubules of epizootic haemorrhagic disease virus using a baculovirus expression system.the formation of virus-specific tubules is one of the most characteristic features in the orbivirus infection cycle, yet little is known about their role in virus replication. the tubuli are composed of a major nonstructural protein, ns1. we have investigated the expression of the ns1-encoding gene of epizootic haemorrhagic disease virus serotype 2 (alberta-strain) by producing a recombinant autographa californica nuclear polyhedrosis virus (acnpv). prior to cloning in the baculovirus transfer v ...19911891959
purification and characterization of the recombinant human aldose reductase expressed in baculovirus system.large quantities of recombinant human aldose reductase were produced using spodoptera frugiperda cells and properties of the enzyme were characterized. direct purification of the recombinant aldose reductase by affinity column chromatography using matrex gel orange a yielded a single 36 kda band, similar in size to the purified human muscle aldose reductase, on a sodium dodecyl sulfate-polyacrylamide gel after silver staining. the isoelectric point of the recombinant enzyme was 5.85 which is ide ...19911905957
purification and characterization of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus.we have constructed the recombinant baculovirus which expresses the human immunodeficiency virus type 1 negative factor (nef) gene. spodoptera frugiperda cells infected with the recombinant virus produced a 27-kda protein which reacted with rabbit antisera raised against a carboxy-terminal synthetic peptide of the nef protein by immunoblot analysis. labeling experiment showed that the recombinant nef protein was myristoylated. the recombinant nef protein was purified to near homogeneity by deae- ...19911909480
characterization of an active, non-myristylated, cytoplasmic form of the lymphoid protein tyrosine kinase pp56lck.pp56lck is a member of the src family of tyrosine kinases mainly expressed in t lymphocytes. src tyrosine kinases have been implicated in the control of cell growth and differentiation in different cell types, but the mechanism of regulation of these enzymes is poorly understood. in order to characterize the distinct species of pp56lck, we have produced high yields of enzymatically active wild type pp56lck using the eukaryotic baculovirus expression system in spodoptera frugiperda insect cells ( ...19911911540
alpha-mannosidase-catalyzed trimming of high-mannose glycans in noninfected and baculovirus-infected spodoptera frugiperda cells (iplb-sf-21ae). a possible contributing regulatory mechanism for assembly of complex-type oligosaccharides in infected cells.incubation of a spodoptera frugiperda (iplb-sf-21ae) cell extract with the oligosaccharide man9glcnac2, the aglucosyl derivative of the glycan that is normally transferred from the dolichol carrier to the relevant asn residue in the nascent protein, results in its trimming to man6glcnac2, an intermediate that is relatively stable to further alpha-d-mannosidase action in these cells. on the other hand, incubation of a similar extract from cells that had been infected for various times with a wild ...19911911772
characterization of a chimeric plasminogen activator consisting of a single-chain fv fragment derived from a fibrin fragment d-dimer-specific antibody and a truncated single-chain urokinase.an mr 57,000 single-chain chimeric plasminogen activator, k12g0s32, consisting of a variable region fragment (fv) derived from the fibrin fragment d-dimer-specific monoclonal antibody ma-15c5 and of a 33-kda (amino acids ala132 to leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-pa-33k) was studied. k12g0s32, secreted by infected spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography a ...19911918077
transcriptional analyses of baculovirus polyhedrin and foreign gene expression relative to baculovirus p10 mrna levels.comparisons have been made between the p10 and polyhedrin mrna levels recovered from spodoptera frugiperda cells infected with autographa californica nuclear polyhedrosis virus (acnpv). in molar terms and from 18 h post-infection (p.i.), the polyhedrin mrna species increased to levels one and a half times to twice as high as the p10 levels. the influence of the polyhedrin leader sequence on the expression of a foreign gene under the control of the polyhedrin promoter was investigated using a ser ...19911919531
baculovirus gp64 gene expression: analysis of sequences modulating early transcription and transactivation by ie1.expression of the baculovirus major envelope glycoprotein gene (gp64) is regulated by transcription from both early and late promoters. to characterize the early promoter and identify sequences involved in the regulation of gp64 early transcription, promoter-reporter gene fusions were generated from the orygia pseudotsugata nuclear polyhedrosis virus gp64 promoter and were analyzed by transient expression in uninfected insect cells. for these analyses, 5' deletion mutations were constructed in t ...19911920618
inhibition of human immunodeficiency virus type 1-induced cell fusion by recombinant human interferons.pretreatment of hela t4 cells with recombinant alpha, beta, or gamma interferon (ifn) was found to significantly inhibit syncytium formation induced by the human immunodeficiency virus type 1 (hiv-1) envelope glycoprotein. all three ifns were found to be potent inhibitors of fusion in a system in which spodoptera frugiperda cells, infected with a baculovirus recombinant expressing the hiv-1 envelope protein, were cocultivated with hela t4 cells. in addition, these ifns were also found to block h ...19911920634
characterization of haemagglutinin-neuraminidase glycoprotein of newcastle disease virus expressed by a recombinant baculovirus.a recombinant baculovirus containing a cdna which encodes haemagglutinin-neuraminidase (hn) of newcastle disease virus (ndv) was constructed. spodoptera frugiperda cells infected with this recombinant virus produced a large amount of hn glycoprotein similar to the authentic hn in size. the recombinant hn glycoprotein was localized on the surface of the infected cells and conserved its haemadsorption and neuraminidase activities. the antigenic properties of the recombinant hn glycoprotein seemed ...19911927050
the single cysteine residue on an alpha family chick liver glutathione s-transferase cl 3-3 is not functionally important.chick liver glutathione s-transferase cl 3-3, expressed using a baculovirus system in spodoptera frugiperda (sf9) cells, contains a single cysteine residue per subunit. this enzyme was modified with iodoacetamide. amino acid analysis indicates that 0.85 +/- 0.10 cysteine residue was modified per enzyme subunit. gst cl 3-3 modified with iodo[14c]acetamide was further digested with trypsin and the isotope-labelled fragments were isolated. the fragment containing the cysteine residue accounts for 5 ...19911930229
generation of recombinant baculovirus via liposome-mediated transfection.baculovirus expression vectors have become a popular method of producing recombinant proteins. production of recombinant virus requires the transfection of both the native viral dna and a transfer plasmid into insect cells where recombination takes place. while several methods of transfecting insect cells exist, we have found liposome-mediated transfection to be highly efficient. here we detail the protocols and medium needed for efficient, simple transfection of spodoptera frugiperda cells.19911931027
high-level secretion of the extracellular domain of the human growth hormone receptor using a baculovirus system.a chemically synthesized gene (hghr-ed) coding for the extracellular domain (ed) of the human growth hormone (hgh) receptor (hghr) was inserted into the genome of autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. spodoptera frugiperda cells infected with the recombinant virus secreted a protein with hgh-binding activity into the medium. the secreted 35-kda protein was purified to near homogeneity. the purified protein exhibited a high binding affinity (kd = 0 ...19911937047
identification of the human platelet gtpase activating protein for the cdc42hs protein.the cdc42hs protein appears to be an isoform of the ras-related gtp-binding protein g25k and is an apparent human homolog of the saccharomyces cerevisiae cell-division-cycle protein, cdc42sc. in this study, we report the identification of a gtpase-activating protein (gap) for cdc42hs from human platelets (designated from here on as cdc42hs-gap). the cdc42hs-gap activity was solubilized from platelet membranes, recovered through successive chromatography steps (the final step being mono-q chromat ...19911939135
evaluation of the role of conserved his and met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase.the 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form his-(x)4-his-(x)4-his-(x)17-his-(x)8-his which represents a potential binding site for non heme iron to the protein. the importance of selected amino acids within this his cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems. after single mutation of each of the 5 his residues at positions 363, 368, 373, 391, and 400 by ser ...19911939225
expression of the tobacco mosaic virus movement protein using a baculovirus expression vector.a cdna clone of the tobacco mosaic virus 30k movement protein (mp) gene was constructed and introduced into an autographa californica nuclear polyhedrosis baculovirus expression vector. infection of spodoptera frugiperda cells with the vector resulted in the synthesis of low levels of mp, which was detected by anti-mp serum as two closely related species of mr approximately 34k and a third species of 32k. the authenticity of the recombinant mp was confirmed by comparison of the protein, on the b ...19911940871
modulation of trehalase activity in two insect cell lines by virus infection and trehalose.trehalase (ec 3.2.1.28), an important glycosidase involved in regulating trehalose levels and metabolic energy in insects, was measured in cell lines from fall army worm, spodoptera frugiperda and salt marsh caterpillar, estigmene acrea, treated with either glucose or trehalose in the presence or absence of tipula iridescent virus (tiv), a cytoplasmic deoxyribovirus. in medium containing 15-35 mm trehalose, both of these cells increased their trehalase activities by 4.5 to 8x the basal levels fr ...19911953800
identification of spliced baculovirus rnas expressed late in infection.previous to this study, the autographa californica multicapsid nuclear polyhedrosis virus (acmnpv) was known to express only one spliced rna (spliced ie1 or ie0). we have conducted an analysis of rna expressed during infection of spodoptera frugiperda cells with acmnpv and have identified a set of five additional spliced rnas expressed late in infection. a reverse transcription-polymerase chain reaction analysis was used to confirm the identification of the ls (late, spliced) rnas. s1 nuclease a ...19911962442
heterologous recombination between autographa californica nuclear polyhedrosis virus dna and foreign dna in non-polyhedrin segments of the viral genome.we used the expression vector system of autographa californica nuclear polyhedrosis virus (acnpv) and spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. we concentrated on the isolation and analysis of heterologous recombinants. the e1 region of human adenovirus type 2 (ad2) was inserted into regions of the acnpv genome which lacked apparent homologies to the polyhedrin region. out of a total of 122 recombinant acnpv plaques, which hybridized to ad2 dna in p ...19911962504
use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.insect cells (spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. when the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the fibra-cel carrier. after infection of the logarithmically growing cells with a recombinant baculovirus (aut ...19911367637
production of recombinant proteins by baculovirus-infected gypsy moth cells.an experimental study was undertaken to evaluate alternative insect cell lines to sf9 [from spodoptera frugiperda (fall armyworm)] for the production of recombinant proteins. insect cell lines from two different organisms were considered: iplb-ldeita (ldeita) from lymantria dispar (gypsy moth) and iplb-hvt1 (hvt1) from heliothis virescens (tobacco budworm). both ldeita and hvt1 produced higher total activity levels of recombinant beta-galactosidase in monolayer culture than sf9 after infection w ...19911367995
microscopic visualization of insect cell-bubble interactions. i: rising bubbles, air-medium interface, and the foam layer.through the use of microscopic, high-speed video technology, the interactions of two suspended insect cell lines, trichoplusia ni (tn-368) and spodoptera frugiperda (sf-9), with air and oxygen bubbles were studied. events such as cell-bubble attachment, cell-bubble collision, cell transport into the foam layer, and trapping of cells in the foam layer are presented and discussed. based on these observations and those in a companion paper (chalmers, j. j.; bavarian, f. biotechnol. prog. 1991, foll ...19911367169
microscopic visualization of insect cell-bubble interactions. ii: the bubble film and bubble rupture.in this paper, the second in the series, the use of a microscopic, high-speed video system to study the interactions of two suspended insect cells strains, trichoplusia ni (tn-368) and spodoptera frugiperda (sf-9), with rupturing bubbles is reported. events such as the adsorption of cells onto the bubble film and the mechanism of bubble rupture were observed. on the basis of these observations and the experimental and theoretical work of other researchers on bubble rupture and cell death as a re ...19911367170
culture of insect cells in helical ribbon impeller bioreactor.an 11-l helical ribbon impeller (hri) bioreactor was tested for the culture of spodoptera frugiperda (sf-9) cells. this impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface-induced babble generation. serum-supplemented and serum-free cultures, using tnmfh and ipl/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h(-1)) to maximum cell densities of 5.5 x 10(6)-6.0 x 10(6) cells. ml(-1) with viability exce ...199118604880
assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus.spodoptera frugiperda insect cells were grown in sf-900 serum-free medium and two kinds of serum-supplemented media (ipl -41 and grace's). the specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees c. the ipl -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/ml) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/ml with the grace's and serum-free media, respectively. in temperature shifdown experiments with a temperat ...199118600874
production of a discrete, heterogeneous population of beta-galactosidase polypeptides using baculovirus expression vectors.gel electrophoresis analysis of immunoprecipitated beta-galactosidase and polyhedrin-beta-galactosidase expressed in spodoptera frugiperda cells infected with recombinant autograph californica nuclear polyhedrosis virus revealed the existence of a population of discrete beta-galactosidase polypeptides. several of the polypeptides observed in the fusion protein expression experiments exhibit a consistent pattern of slightly greater molecular weight when compared to the nonfusion beta-galactosidas ...199218601031
optimization of the production of full-length rcd4 in baculovirus-infected sf9 cells.the baculovirus-insect cell system is reliable in expressing a variety of recombinant proteins. a recombinant baculovirus encoding the full length human cd4 has been used to infect spodoptera frugiperda 9 cells in 6-l-airlift fermentors. the procedured described in this report permitted a 6.5-fold enhancement of rcd4 expression as compared to standard procedures previously published. the increase of rcd4 expression on the cell surface was achieved by using the following steps: (1) optimal seedin ...199218601106
a structured dynamic model for the baculovirus infection process in insect-cell reactor configurations.a mathematical description of the infection of insect cells with baculovirus in a continuously operated reactor configuration is presented. the reactor configuration consists of one bioreactor in which insect cells (spodoptera frugiperda) are grown followed by one or two bioreactors in which cells are infected by a baculovirus (autographa californica nuclear polyhedrosis virus). it was demonstrated that the so-called passage effect is responsible for the observed difference in run time between a ...199218601149
baculovirus expression vectors.this chapter aims to provide the reader with the experimental protocols required to produce a baculovirus containing the gene of interest, including transfection of spodoptera frugiperda cells and the screening of progeny virus by plaque assay. in addition, an outline of the biochemical methods routinely used to characterize the expressed protein product within the baculovirus system will be given. throughout the experimental procedures, areas that researchers generally find technically difficul ...199221390722
6,10,14-trimethylpentadecan-2-one: a bermuda grass phagostimulant to fall armyworm (lepidoptera: noctuidae).a phagostimulant, 6,10,14-trimethylpentadecan-1-one (phytone), was isolated and identified from bermuda grass,cynodon dactylon (l.). the phagostimulant activity of this isoprenoid ketone was established from bioassays of fall armyworm larvae,spodoptera frugiperda (j.e. smith). larvae displayed increased body mass accumulation as well as preference to diet supplemented with this molecule. neonate larvae fed diet supplemented with chromatographic isolates of phytone-containing fractions from six b ...199224253874
a sensitive model system for in vivo monitoring of baculovirus gene expression in single infected insect cells.we have developed a fast and sensitive system for the in vivo analysis of gene expression in baculovirus infected lepidopteran insect cells. a recombinant baculovirus containing a luciferase gene from the click beetle, pyrophorus plagiophthalamus, under transcriptional regulation of the polyhedrin gene promoter of autographa californica nuclear polyhedrosis virus (acnpv) was used to infect a spodoptera frugiperda cell line. recombinant luciferase could be monitored by luminometry in real-time wi ...19921368236
optimization of protein-production by the baculovirus expression vector system in shake flasks.shake flasks were successfully employed for the cultivation of spodoptera frugiperda (sf-9) insect cells and for the production of beta-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. the culture doubling time and maximal cell density were 20 h and 5 x 10(6) cells/ml respectively. the optimal liquid volumes for flasks rotating at 100 rpm were 25-40% of the flask total volume. enzyme production (about 600 mg/l) was best at a multiplicity of infectio ...19921368502
effect of glycosylation on properties of soluble interferon gamma receptors produced in prokaryotic and eukaryotic expression systems.we investigated the influence of glycosylation on solubility, chromatographic behavior and resistance to heat- and chaotrope-dependent denaturation and proteolytic digestion of three recombinant human interferon gamma receptors produced in escherichia coli, spodoptera frugiperda and chinese hamster ovary cells. the proteins produced in the eukaryotic expression systems were glycosylated, carrying different, heterogeneous carbohydrate moieties. they were assayed fully glycosylated and after remov ...19921368793
baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion.we have devised a simple and efficient baculovirus expression vector system to evaluate insect tissue culture cells for their capacity to express, glycosylate and secrete foreign proteins. a truncated placental alkaline phosphatase (seap) gene was inserted into the autographa californica nuclear polyhedrosis virus (acmnpv) genome under the transcriptional control of the polyhedrin gene promoter. production levels, glycosylation, and secretion of the recombinant protein were examined in trichoplu ...19921368794
trypanosoma cruzi flagellar repetitive antigen expression by recombinant baculovirus: towards an improved diagnostic reagent for chagas' disease.we constructed a recombinant baculovirus that expressed part of a trypanosoma cruzi flagellar repetitive antigen (fra). both cell- associated and secreted forms of recombinant fra were detected in cultures of virus-infected spodoptera frugiperda (sf9) cells. these forms show a complex pattern after polyacrylamide gel electrophoresis and western blot analysis using either an anti-fra rabbit serum or human chagasic sera. competitive western-blot experiments revealed that all bands react with the s ...19921369025
recombinant beta-galactosidase production in serum-free medium by insect cells in a 14-l airlift bioreactor.spodoptera frugiperda (sf9) insect cells were successfully cultured in serum-free medium in a 14-l airlift bioreactor. cell densities as high as 1 x 10(7) cells/ml were achieved with specific growth rates of approximately 0.0286 h-1 (doubling time of 24 h). this system was also used to demonstrate the expression of a reported gene, beta-galactosidase (beta-gal), when cells were infected with a recombinant baculovirus. approximately 0.33 mg of beta-gal/ml (i.e., 104,000 units/ml) of medium were o ...19921369040
recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus.spodoptera frugiperda (iplb-sf-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (cat) in serum-free medium (sf-900) and two serum-supplemented media (ipl-41 and grace's). in temperature-shift experiments (cell growth at 33 degrees c followed by virus replication at 27 degrees c 3-4 days later), virus and cat production were much poorer in the serum-free medium than in serum-supplemented med ...19921369167
screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system.eight cell lines derived from the insects spodoptera frugiperda, trichoplusia ni, mamestra brassicae, and estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus autographa californica multiple nuclear polyhedrosis virus (acmnpv). production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. cell density was found to be an important fa ...19921369220
effects of oxygen on recombinant protein production by suspension cultures of spodoptera frugiperda (sf-9) insect cells.spodoptera frugiperda (sf-9) insect cells have been grown in serum-free medium in 250-ml spinner flasks. the maximum cell density obtained in these cultures was dependent on the aeration rate of the culture. similar yields of uninfected cells were obtained when cultures were stirred in spinner flasks at 80 rev min-1 and in a 4-1 stirred-tank bioreactor and the dissolved oxygen in the bioreactor was controlled at 20% of air saturation. cells were infected with a recombinant baculovirus at differe ...19921369406
purification and characterization of recombinant xenopus poly(a)(+)-binding protein expressed in a baculovirus system.the poly(a)(+)-binding protein (pabp) is a highly conserved protein that binds to the poly(a)+ tail of mrnas. pabp has been shown to regulate message stability and translational efficiency, yet the mechanisms remain unknown. to facilitate further dissection of the functions of this protein, we have expressed and purified xenopus pabp using a baculovirus expression system. at 48 h after infection of insect spodoptera frugiperda (sf9) cells with recombinant virus, approx. 3% of cell protein was pa ...19921280105
bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae.bacterial luciferase, derived from a fusion of the luxa and luxb genes of vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. the coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. these vectors contained the beta-galactosidase indicator gene under control of immediate early (ie1), early (etl), or very late (p10) promoters and a cloning site for inserting the fus ...19921308004
herpes simplex virus type 1 origin-dependent dna replication in insect cells using recombinant baculoviruses.the minimal set of seven herpes simplex virus type 1 (hsv-1) genes required for viral origin-dependent dna synthesis was previously identified using a transient replication assay in a mammalian cell line permissive for hsv-1 growth. we have constructed recombinant baculoviruses which efficiently express the products of each of these seven genes in infected spodoptera frugiperda (sf) insect cells. when sf cells were transfected with a plasmid containing a functional hsv-1 origin of replication, a ...19921311360
in vitro transactivation of baculovirus early genes by nuclear extracts from autographa californica nuclear polyhedrosis virus-infected spodoptera frugiperda cells.nuclear extracts, prepared from autographa californica nuclear polyhedrosis virus-infected spodoptera frugiperda cells during a time course of infection, were analyzed for activation of early gene transcription and for late gene transcription. the templates used in the in vitro transcription assays contained promoters for baculovirus genes that have been classified as immediate early, delayed early, and late. the promoters were derived from the baculovirus 39k, p26, gp64, and dna polymerase gene ...19921316463
physical and functional interaction of human cytomegalovirus dna polymerase and its accessory protein (icp36) expressed in insect cells.expression of the human cytomegalovirus (hcmv) (ad169) dna polymerase gene under the control of the polyhedrin promoter of autographa californica nuclear polyhedrosis virus in spodoptera frugiperda (sf9) cells has provided a source of highly active cmv dna polymerase. in extracts from cmv-infected cells, the cmv dna polymerase is found strongly associated with an additional polypeptide, icp36. this protein has been identified as the cmv homolog of the herpes simplex virus type 1 ul42 gene produc ...19921318399
expression of the nonstructural proteins of sindbis virus in insect cells by a baculovirus vector.the genome of sindbis virus encodes the polypeptides that are required for the replication and transcription of the virus rna in infected cells. these polypeptides are translated as a polyprotein that is co- and post-translationally cleaved by an autoproteinase to give rise to four polypeptides designated nsp1, nsp2, nsp3 and nsp4. we have initiated a study of the functions of these proteins by expressing them in the autographa californica baculovirus polyhedrin expression system. spodoptera fru ...19921320794
baculovirus expression of pestivirus non-structural proteins.bovine viral diarrhoea virus (bvdv) belongs to the pestivirus group, a genus within the flaviviridae family. it possesses a positive-sense ssrna genome with a single large open reading frame (orf) encoding about 4000 amino acids. here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. the 3'-terminal 60% of the bvdv orf was cloned into a plasmid transfer vector which was then used to construct a ...19921321220
characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.prolyl 4-hydroxylase (ec 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. the enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. molecular cloning of the catalytically important alpha subunit has identified two types of cdna clone due to mutually exclusive alternative splicing. the beta subunit is a highly unusual multifunctio ...19921323838
site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in spodoptera frugiperda insect cells.protein disulphide isomerase (pdi) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. it has two -cys-gly-his-cys- sequences which represent two independently acting catalytic sites of pdi activity. we report here on the expression in baculovirus vectors of various mutant pdi/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in spodoptera frugiperda insect cells. when either one or ...19921327760
expression in insect cells and immune reactivity of a 28k tegument protein of human cytomegalovirus.the gene encoding the highly antigenic 28k (pp28) tegument phosphoprotein of human cytomegalovirus (hcmv) was expressed in insect cells utilizing a recombinant baculovirus. the mature intracellular form of the recombinant-derived pp28 had mobility on sds-polyacrylamide gels similar to that of native pp28 from hcmv strain towne-infected human foreskin fibroblasts (hffs). in vitro labelling of recombinant autographa california nuclear polyhedrosis virus-infected spodoptera frugiperda cells or of h ...19921328492
assembly of functional gabaa receptors in insect cells using baculovirus expression vectors.we have constructed recombinant baculoviruses containing cdnas encoding either the alpha 1- or the beta 1-subunit of the bovine gabaa receptor. in spodoptera frugiperda (iplb-sf-21) cells infected with recombinant virus expressing either the alpha 1- or beta 1-subunit, or in cells co-infected with both viruses, functional gabaa receptors were detected by whole-cell electrophysiological recordings. the threshold for the responses mediated by the homo-oligomeric channels (alpha- or beta-) was 2-3 ...19921330062
baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific dna-binding behavior with the 1,25-dihydroxyvitamin d3 receptor.the baculovirus genetic expression system has been used to produce murine retinoic acid receptor (rar) type gamma in spodoptera frugiperda insect cells and manduca sexta insect larvae. a hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of rar gamma per mg of protein. the cytosolic preparation from infected insect cells exhibited an equilibrium dis ...19921332041
stimulation of thymidine kinase activity in baculovirus infected cells is not due to a virus-coded enzyme.a polyhedrin-positive recombinant autographa californica nuclear polyhedrosis virus (acnpv) carrying a herpes simplex virus thymidine kinase gene under the control of the synxiv promoter, a fusion of synthetic and linker-modified polyhedrin promoters, has been constructed. when this recombinant baculovirus was used to infect a variant of spodoptera frugiperda cells deficient in thymidine kinase (tk-), a high level of tk activity was detected. these results, in conjunction with the demonstration ...19921333755
cells transfected with human interleukin 6 cdna acquire binding sites for the hepatitis b virus envelope protein.earlier studies revealed that human interleukin 6 (il-6) contains recognition sites for the hepatitis b virus (hbv) envelope (env) protein, and that il-6 and anti-il-6 antibodies, respectively, inhibited the interaction of cells expressing a receptor for hbv with the pres(21-47) segment of the hbv env protein, encompassing the complementary attachment site for il-6. this suggested that il-6 mediates hbv-cell interactions. we report that: (a) chinese hamster ovary cells transfected with human il- ...19921334115
activating transcription factor-2 dna-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases.recent studies have detailed the ability of activating transcription factor-2 (atf-2) to mediate adenoviral e1a stimulation of gene expression; however, an endogenous regulator for the transcriptional activity of this protein has not been described. to characterize the regulation of atf-2 activity, we have expressed full-length and truncated peptides corresponding to various regions of the atf-2 protein in bacteria and the baculovirus insect cell system. bacterially expressed truncated (350-505) ...19921337144
cloning and expression of a chick liver glutathione s-transferase cl 3 subunit with the use of a baculovirus expression system.glutathione s-transferase cl 3 subunits purified from 1-day-old-chick livers were digested with achromobacter proteinase i and the resulting fragments were isolated for amino acid sequence analysis. an oligonucleotide probe was constructed accordingly for cdna library screening. a cdna clone of 1342 bases, pgcl301, encoding a protein of 26209 da was isolated and sequenced. including conservative substitutions, this protein has 75-79% sequence similarity to other alpha family glutathione s-transf ...19921339283
alzheimer beta/a4-amyloid precursor protein: evidence for putative amyloidogenic fragment.recombinant baculovirus was used to overexpress human alzheimer beta/a4-amyloid precursor protein (app) in spodoptera frugiperda (sf9) cells. lysates of these cells were then analyzed for the presence of carboxyl-terminal fragments of app by an immunoblotting assay using either an antibody against the app cytoplasmic domain (rabbit anti-human 695app645-694) or an antibody against the amino terminus of beta/a4-amyloid (rabbit anti-human 695app586-606). anti-human 695app645-694 identified app holo ...19921345769
detection of protein kinase homologues and viral rna-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds rna-activated 68,000-da protein kinase.the p68 protein kinase (referred to as p68 based on its m(r) of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by dsrnas. the kinase is under tight controls in virus-infected cells since once activated, it phosphorylates its natural substrate eukaryotic initiation factor 2 (elf-2), leading to potential limitations in functional elf-2 and decreases in protein synthesis initiation. to further delineate the molecular mechanisms underlying kinase re ...19921360180
bovine rotavirus segment 5 protein expressed in the baculovirus system interacts with zinc and rna.the cdna sequence of genomic segment 5 of bovine rotavirus (rf strain) has been inserted into baculovirus transfer vectors, downstream of the polyhedrin promotor. recombinant baculoviruses containing gene 5 were selected and the protein was expressed to high yields in spodoptera frugiperda cells. the recombinant protein was inoculated into rabbits and mice to produce specific hyperimmune antisera. the polyclonal antiserum reacted with a protein in rotavirus-infected ma104 cells and with a protei ...19921379624
presentation of hepatitis b virus pres2 epitope on bluetongue virus core-like particles.a chimeric protein containing most of the hepatitis b virus pres2 region (amino acid residues 1-48) upstream to, and colinear with the amino-terminus of bluetongue virus vp7 protein (pres2-vp7) was expressed by a recombinant autographa californica nuclear polyhedrosis virus (acnpv). the chimeric protein formed btv core-like particles (clps) in spodoptera frugiperda cells only when the cells were coinfected with this recombinant virus and a recombinant baculovirus that expresses unmodified vp7 an ...19921381538
potential role of monocyte chemoattractant protein 1/je in monocyte/macrophage-dependent iga immune complex alveolitis in the rat.we have examined the role of monocyte chemoattractant protein 1 (mcp 1) in the pathogenesis of monocyte/macrophage-dependent iga immune complex alveolitis in the rat. rat mcp 1 was cloned and expressed in order to facilitate analysis of its function in rat models of human disease. a cdna library was constructed from rat pulmonary artery endothelial cells stimulated with tnf-alpha. the cdna library was screened with synthetic oligonucleotide probes based on the recently published rat mcp 1 cdna s ...19921387671
expression, purification, and functional characterization of adenovirus 5 and 12 e1a proteins produced in insect cells.the 12 s and 13 s e1a cdnas from both the adenovirus (ad) nononcogenic type 5 and the oncogenic type 12 were overexpressed in an insect cell/baculovirus system. upon infection of spodoptera frugiperda cells, the production of e1a proteins reached a level of about 15 micrograms/10(6) cells. the e1a proteins are highly soluble and apparently are processed authentically. they are readily recognized by various antibodies and display phosphorylation patterns similar to those of e1a proteins synthesiz ...19921387752
purification and characterization of a low m(r) gtp-binding protein, ram p25, expressed by baculovirus expression system.the ram gene was isolated from rat megakaryocyte cdna library with an oligonucleotide probe which is specific for a low m(r) gtp-binding proteins c25kg purified from human platelets. its gene product (ram p25) is a monomeric 25-kda guanine nucleotide-binding protein. the protein was expressed by using baculovirus transfer vector, pacym1, which allowed the production at a high level of soluble recombinant ram p25 in spodoptera frugiperda (sf9) cells under the control of polyhedrin promoter. the e ...19921390922
characterization of rabies virus glycoprotein expressed by recombinant baculovirus.a cdna of the glycoprotein (g protein) gene of rabies virus nishigahara strain was cloned and inserted into a baculovirus genome under the control of the polyhedrin promoter. infection of spodoptera frugiperda cells with this recombinant virus produced a large quantity of new protein instead of the parental polyhedrin protein. by immunofluorescent and immunoblotting analyses, the recombinant protein was antigenically similar to the authentic g protein. its molecular mass estimated by sodium dode ...19921413990
purification of pancreatic cholesterol esterase expressed in recombinant baculovirus-infected sf9 cells.a cdna clone encoding the entire coding sequence of rat pancreatic cholesterol esterase (bile salt-stimulated lipase) was subcloned into the baculovirus transfer vector pvl1392 and used to co-transfect spodoptera frugiperda (sf9) insect cells with wild-type autographa californica nuclear polyhedrosis virus (acnpv) dna. two recombinant proteins (m(r) 74 kda and 64 kda) reactive with anti-cholesterol esterase igg were produced and secreted by the infected sf9 cells in large quantities in a time-de ...19921422214
high-level production of human alpha- and beta-globins in insect cells.high-level production of human alpha- and beta-globins in cultured spodoptera frugiperda (sf-9) cells infected with recombinant baculoviruses is described. the expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. two recombinant baculoviruses for alpha-globin, h alpha and h beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mrna coding region in h beta ...19921422217
production, purification, and characterization of human recombinant il-8 from the eucaryotic vector expression system baculovirus.the cdna for the human chemotactic interleukin, il-8, was subcloned from a bacterial source into the eucaryotic vector expression system baculovirus. recombinant human il-8 (rhil-8) was synthesized and secreted from sf9 cells derived from spodoptera frugiperda following infection of a recombinant virus harboring the full-length il-8 structural gene. infected sf9 cells produced rhil-8 in a range from 0.5 to 2.0 mg of rhil-8/liter of postinfection cell culture media. the recombinant interleukin wa ...19921422225
recombinant laminin b1 chains exhibit intact short-arm domains but do not form oligomeric molecules.the human laminin b1 chain has been produced in the baculovirus expression system in sufficient amounts for biochemical and functional studies. a full-length cdna, which was constructed of four partially overlapping clones and verified by in vitro transcription and translation to be functional, was cloned into the transfer vector pvl1392 behind the polyhedrin promoter. the recombinant construct was incorporated by in vivo homologous recombination into the genome of the wild-type baculovirus, aut ...19921425664
expression of cauliflower mosaic virus orf ii in a baculovirus system.the cauliflower mosaic virus orf ii encoding the aphid transmission factor (atf) was mutagenized to introduce a bamhi restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (autographa californica nuclear polyhedrosis virus). all recombinant viruses tested in spodoptera frugiperda (sf21) cells expressed a protein of about 18 kd which comigrated in page with atf from infected plants. western blotting using an oligopeptide antiserum to atf co ...19921428752
the identification and characterization of a gdp-dissociation inhibitor (gdi) for the cdc42hs protein.the ras-related protein, cdc42hs, is a 22-kda gtp-binding protein which is the human homolog of a saccharomyces cerevisiae yeast-cell-division cycle protein. in attempting to isolate and biochemically characterize mammalian proteins capable of regulating various activities of cdc42hs, we have identified an activity in bovine brain cytosol which effectively inhibits the dissociation of [3h]gdp from the platelet- or the spodoptera frugiperda-expressed cdc42hs protein. the purification of this acti ...19921429634
sequence evolution in mitochondrial ribosomal and nd-1 genes in lepidoptera: implications for phylogenetic analyses.a 2,256-bp sequence of the mitochondrial genome of a lepidopteran (spodoptera frugiperda) contains trnas for valine and leucine, the 16s rrna, and three-quarters of the nd-1 presumptive protein-coding gene. a 64-bp stretch of unknown function was located between the rrna and leucine trna. sequence divergence in the 16s rrna obtained from alignment with published insect sequences is consistent with phylogenetic hypotheses, in that diptera and lepidoptera are more closely related to each other (24 ...19921435234
expression and secretion of japanese encephalitis virus nonstructural protein ns1 by insect cells using a recombinant baculovirus.the nonstructural protein ns1 of japanese encephalitis virus (jev) was expressed at a high level under the control of the polyhedrin promoter in spodoptera frugiperda (sf9) insect cells using a recombinant baculovirus. recombinant ns1 was designed to contain its natural signal sequence at its n-terminus and no c-terminal hydrophobic domain that could act as a membrane anchor. this recombinant protein exhibited similar size to native ns1 expressed in aedes albopictus (c6/36) insect cells infected ...19921448926
studies on processing, particle formation, and immunogenicity of the hiv-1 gag gene product: a possible component of a hiv vaccine.antigens in a particulate conformation were shown to be highly immunogenic in mammals. for this reason, the particle forming capacity of derivatives of the hiv-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. moreover, p55 gag was expressed in combination with ...19921456888
analysis of soluble human and mouse interferon-gamma receptors expressed in eukaryotic cells.the extracellular domains of the human and mouse interferon-gamma receptors were produced in insect spodoptera frugiperda cells infected with recombinant baculoviruses and in mammalian chinese-hamster-ovary cells. the receptors expressed in both systems are secreted into the culture medium. their signal peptides are cleaved off and the proteins show heterogeneity in glycosylation which, however, does not affect the capacity to bind interferon gamma or specific antibodies. the soluble mouse recep ...19921459135
studies on the interaction of alpha subunits of gtp-binding proteins with beta gamma dimers.the interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(g)-protein alpha subunits, a recombinant bv alpha i3, made in sf9 spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte gs-type gtp-binding protein, and activated by naf/alcl3, or unpurified as found in a natural membrane, were studied. the beta gamma dimers used were from bovine rod outer segments (r ...19921459143
distinct signals in human immunodeficiency virus type 1 pr55 necessary for rna binding and particle formation.the human immunodeficiency virus type 1 (hiv-1) gag gene product pr55 self-assembles to form virus-like particles when expressed in spodoptera frugiperda cells using recombinant baculoviruses. the particles resemble immature hiv and are released from the infected cell into the culture medium. using this system we have progressively truncated the gag open reading frame from the c terminus and examined each deleted gag protein for its particle-producing capability. we show that deletion of pr6 and ...19921469349
transient expression assay in a baculovirus system using firefly luciferase gene as a reporter.transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses bombyx mori nuclear polyhedrosis virus (bmnpv) and autographa californica nuclear polyhedrosis virus (acnpv) in insect cells of bombyx mori and spodoptera frugiperda, respectively. dna sequences encoding luciferase (luc) of the firefly photinus pyralis was successfully employed in the expression assay as a reporter gene. recombinant plasmids were constructed containing the luc gene ...19921475909
characterization of baculovirus-expressed hemagglutinin and fusion glycoproteins of the attenuated measles virus strain aik-c.with measles virus cdna of the avirulent vaccine strain aik-c, two cdnas of h or f genes were amplified by the polymerase chain reaction. the amplified cdnas were inserted respectively to the baculovirus transfer vector pacym1 derived from the nuclear polyhedrosis virus of autographa californica (acnpv). after co-transfection of the transfer vectors with acnpv dna to spodoptera frugiperda cells, recombinant baculoviruses were screened by plaque assay, and the viruses containing h-cdna or f-cdna ...19921476005
human preproendothelin-1 is converted into active endothelin-1 by baculovirus-infected insect cells.to investigate biochemical and biological parameters involved in preproendothelin-1 (preproet-1) maturation we infected spodoptera frugiperda (sf21) cells with a suitable engineered baculovirus vector carrying the cdna encoding the entire human 212 amino acids precursor. culture supernatants were tested by ria using an anti-et-1 serum, et-1-like immunoreactive material (irm) was detected in the infected sf21 cells but not in control, wild-type or mock-infected cells. fractionation of the culture ...19921497664
distinct n-glycan fucosylation potentials of three lepidopteran cell lines.the fucosyltransferase activities of three insect cell lines, mb-0503 (from mamestra brassicae), bm-n (from bombyx mori) and sf-9 (from spodoptera frugiperda), were investigated and compared with that of honeybee venom glands. cell extracts and venom gland extracts were incubated with gdp-[14c]fucose and glycopeptides isolated from human igg and from bovine fibrin. the labeled oligosaccharide products were released by peptide-n4-(n-acetyl-beta-glucosaminyl)asparagine amidase a, fluorescence mark ...19921499571
site-specific mutagenesis of the 35-kilodalton protein gene encoded by autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.the gene encoding the 35-kda protein (35k gene) located within the ecori-s genome fragment of autographa californica nuclear polyhedrosis virus (acmnpv) is transcribed early in infection. to examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the acmnpv genome. in spodoptera frugiperda (sf21) culture, deletion of the 35k gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. ...19921501287
overexpressed full-length human bcl2 extends the survival of baculovirus-infected sf9 insect cells.full-length and truncated human bcl2 lacking the entire c-terminal hydrophobic domain have been overexpressed in spodoptera frugiperda insect cells with the baculovirus expression system. immunoblot analysis with bcl2-specific antibodies revealed that both full-length and truncated bcl2 are expressed as multiple immunoreactive species, suggesting posttranslational modifications. the expression of the full-length but not the truncated bcl2 extended the survival of baculovirus-infected cells by pr ...19921502141
a synthetic early promoter from a baculovirus: roles of the tata box and conserved start site cagt sequence in basal levels of transcription.many baculovirus early genes and insect genes transcribed by rna polymerase ii have a conserved transcription start site sequence (cagt) located downstream of a consensus tata box. to examine the functions and interactions of these two motifs in initiating accurately positioned basal transcription, a 43-nt synthetic promoter was synthesized from the tata box and start site sequences of the gp64 early promoter from the orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (opmnpv). the synt ...19921519359
site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione s-transferase 3-3.rat liver glutathione s-transferase (gst) 3-3 is composed of two identical subunits, each containing three cysteine residues, cys-86, cys-114 and cys-173. we have shown previously that cys-86 is not involved in the enzymic activity of gst 3-3 [hsieh, huang, chen, lai & tam (1991) biochem, j. 278, 293-297]. at 50 degrees c, iodoacetamide can inactivate the enzyme by modifying cys-86 and cys-114. cys-114 can be protected against iodoacetamide inhibition by s-(dinitrophenyl)glutathione. site-direct ...19921520269
expression of two s-ribonucleases of petunia inflata using baculovirus expression system.we have previously shown that three petunia inflata s-proteins, products of the multiallelic s-gene of the self-incompatibility system, are ribonucleases. here we report the expression of cdnas for two of these s-proteins using the baculovirus expression system. s2- and s3-proteins were found in both supernatants and lysates of spodoptera frugiperda cells infected with recombinant baculoviruses. both recombinant s-proteins contained glycosylated (25 kd) and nonglycosylated (23 kd) forms. recombi ...19921520311
hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modifications between the large and small forms.hepatitis delta virus (hdv) encodes only one protein, the hepatitis delta antigen (hdag). two forms of hdag, a large (27 kda) and a small (24 kda) one, participate in the various steps of hdv replication. to further understand the properties of hdag, we have constructed recombinant baculoviruses and expressed both forms of the hdag in insect cells. the gene encoding hdag was placed under the control of the polyhedrin promoter of autographa californica nuclear polyhedrosis virus (acnpv) by homolo ...19921529540
expression of the nucleocapsid protein of dugbe virus and antigenic cross-reactions with other nairoviruses.the small (s) rna segment of dugbe (dug) virus (nairovirus, bunyaviridae) encodes a single protein, the nucleocapsid (n) protein, of m(r) 49.4 kda. cdna derived from the complete coding region for the n protein was cloned into autographa californica nuclear polyhedrosis virus (acnpv) under control of the polyhedrin promoter and used to infect spodoptera frugiperda insect cells. western blotting analysis using monoclonal antibodies demonstrated the production of dug n protein in the infected cell ...19921529645
baculovirus-mediated expression and functional characterization of human nadph-p450 oxidoreductase.human nadph-p450 oxidoreductase (or) is an intrinsically membrane-bound flavoprotein that serves to transfer electrons from nadph to cytochrome p450. or is also involved in the metabolic activation of chemotherapeutic alkylating agents. the human or cdna was engineered into baculovirus and the recombinant virus was used to infect spodoptera frugiperda (sf9) cells. approximately 3.3% of total protein of infected cells was human or. the enzyme was purified by ion exchange and affinity chromatograp ...19921536559
expression of androgen receptor in insect cells. purification of the receptor and renaturation of its steroid- and dna-binding functions.a full-length rat androgen receptor cdna was used to produce a recombinant baculovirus (acrar) by homologous recombination. spodoptera frugiperda (sf9) cells infected with this virus expressed a 110-kda polypeptide that amounted up to about one-third of total cell protein. studies with ar antibodies confirmed that this protein was indeed rar. only a minor portion of the recombinant ar was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 m guanidine h ...19921537872
high-level synthesis and fate of acetylcholine in baculovirus-infected cells: characterization and purification of recombinant rat choline acetyltransferase.rat choline acetyltransferase (chat) has been expressed at a high level in spodoptera frugiperda sf9 cells using a baculovirus expression system. a cdna containing the coding sequence for chat was inserted into the transfer vector pacym1 to yield the recombinant vector pacym1/chat. sf9 cells were then coinfected with pacym1/chat and the wild-type autographa californica virus. one recombinant virus particle, containing the cdna for chat, was selected that expressed a protein of 68.5 kda. forty ho ...19921548478
biophysical and biochemical properties of baculovirus-expressed camv p1 protein.cauliflower mosaic virus (camv) gene i encodes a protein (p1) that has been implicated in the control of virus movement in infected plants. to assist in the characterization of the mechanism of action of p1, gene i has been expressed efficiently in spodoptera frugiperda (sf) cells using recombinant baculovirus. control of the expression of camv gene i by the polyhedrin late promoter in the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) resulted in very high levels of p1 ac ...19921549910
expression of prostaglandin endoperoxide synthase-1 in a baculovirus system.a cdna coding for ovine prostaglandin endoperoxide (pgh) synthase-1 was used to construct a recombinant baculovirus which was expressed in spodoptera frugiperda (sf9) insect cells. two proteins reactive with anti-pgh synthase antibody were produced. a larger protein (mr = 72,000) coelectrophoresed with native enzyme; a smaller, more abundant protein (mr = 66,000) was unglycosylated enzyme. about 90% of both the immunoreactivity and the cyclooxygenase activity were present in a low speed (10(5) g ...19921567411
expression and transport into mitochondria of bovine cytochrome p-450(scc) in insect cells using the baculovirus expression system.bovine cytochrome p-450(scc) introduced with the baculovirus host vector system was found to be expressed in spodoptera frugiperda cells. cell fractionation analysis indicated that the p-450(scc) expressed as the precursor form was transported into mitochondria and converted to a mature form. however, this form did not exhibit definite activity for cholesterol side chain cleavage. these findings suggest that most of the p-450(scc) expressed by this system is an inactive protein within mitochondr ...19921567449
synapsin iia: expression in insect cells, purification, and characterization.synapsin iia belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. in order to examine the biochemical properties of synapsin iia, and ultimately its physiological function, purified protein is required. since attempts to purify significant quantities of synapsin iia, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin iia by util ...19921567872
the nadp-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase is not expressed in spodoptera frugiperda cells.the insect cell line derived from spodoptera frugiperda (sf9) does not express the activities of the trifunctional nadp-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. the lack of synthetase activity was confirmed by the inability to incorporate radiolabeled formate into nucleotides. the cells express, instead, a mg2+ and nad-dependent bifunctional methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohy ...19921569082
differential factor binding at the promoter of early baculovirus gene pe38 during viral infection: gata motif is recognized by an insect protein.regulatory elements interacting with dna-binding proteins have been investigated in the promoter sequence of the early pe38 gene in the autographa californica nuclear polyhedrosis virus (acnpv). a gata motif located 50 nucleotides upstream of the pe38 transcriptional start site is recognized differentially in the course of infection. as demonstrated by footprint and gel mobility shift assays, the gata sequences ttatct are protected by nuclear extracts from uninfected spodoptera frugiperda cells ...19921583720
isopentenoid synthesis in embryonic drosophila cells: prenylated protein profile and prenyl group usage.it has been established that vertebrates and yeasts modified a unique subset of polypeptides with farnesyl and geranylgeranyl residues. this observation has been extended to drosophila kc cells. [3h]mevalonate was incorporated into 54 kc cell peptides (18-92 kda). as reported for mammalian cells, most of the labeled peptides had molecular weights between 21 and 27 kda. c18 radio-hplc tryptic digest profiles for delipidized, [3h]mevalonate-labeled (a) insect (drosophila and spodoptera frugiperda) ...19921586169
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