| structures of the n-linked oligosaccharides of the membrane glycoproteins from three lepidopteran cell lines (sf-21, izd-mb-0503, bm-n). | the primary structures of the asn-linked carbohydrate chains isolated from membrane glycoproteins of the three insect cell lines mamestra brassicae (mb-0503), bombyx mori (bm-n), and spodoptera frugiperda (sf-21) have been determined. tryptic glycopeptides derived from the membrane fraction were digested with peptide-n-glycanase a. the resulting oligosaccharides were reductively aminated with 2-aminopyridine and identified by two-dimensional hplc mapping in combination with exoglycosidase digest ... | 1994 | 8311447 |
| comparative recombinant protein production of eight insect cell lines. | a recombinant autographa californica baculovirus expressing secreted alkaline phosphatase (seap) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived from spodoptera frugiperda, trichoplusia ni, mamestra brassicae and estigmene acrea. because cell density was found to influence protein production, seap production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. on a per cell basis, the t. ni- ... | 1993 | 8314732 |
| identification and immunocharacterization of a 25k structural protein in autographa californica nuclear polyhedrosis virus (acmnpv). | a 25k protein was a major component of the triton x-100 solubilized fraction prepared for the isolation of nuclear matrices from spodoptera frugiperda cells infected with autographa californica nuclear polyhedrosis virus (acmnpv). polyclonal antisera to p25 were produced in rabbits and used in the characterization of p25. western blots of proteins probed with the p25 antiserum demonstrated the presence of the p25 in both the polyhedral-derived and extracellular viral phenotypes. using an indirec ... | 1993 | 8317143 |
| development of baculovirus triple and quadruple expression vectors: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells. | baculovirus multiple gene transfer vectors pacab3 and pacab4 have been developed to facilitate the insertion of three or four foreign genes respectively into the autographa californica nuclear polyhedrosis virus (acnpv) genome by a single co-transfection experiment. the pacab3 vector contains a polyhedrin promoter and two p10 promoters on either side of the polyhedrin promoter but in opposite orientations. the pacab4 vector has an additional polyhedrin promoter in opposite orientation to the fir ... | 1993 | 8385313 |
| baculovirus-expressed herpes simplex virus type 2 glycoprotein d is immunogenic and protective against lethal hsv challenge. | herpes simplex virus type 2 glycoprotein d (gd2) was cloned and expressed in the baculovirus-spodoptera frugiperda system. milligram quantities of glycoprotein were recovered from suspension culture and subjected to purification by ion-exchange and immunoaffinity chromatography. the resultant purified gd existed as a homogeneous 57,500 mw monomeric species demonstrating reactivity with anti-gd monoclonal antibodies including those directed at a non-sequential neutralizing epitope of gd. immuniza ... | 1993 | 8385842 |
| non-homologous recombination between adenovirus and acnpv dna fragments in cell-free extracts from insect spodoptera frugiperda nuclei. | in previous work, we have developed a cell-free system from nuclear extracts of hamster cells to study the mechanism of integrative recombination between adenovirus type 12 (ad12) dna and hamster cell dna (jessberger et al., 1989; tatzelt et al., 1992). we have also demonstrated that in insect cells the left terminal fragment of ad2 dna can insert by non-homologous recombination into the 32.6 to 34.5 map unit (ecori-o) fragment and into other segments of autographa californica nuclear polyhedros ... | 1993 | 8317144 |
| purification and characterization of recombinant histidine-tagged human platelet 12-lipoxygenase expressed in a baculovirus/insect cell system. | a baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cdna, under control of the polyhedrin promoter, was constructed. recombinant 12-lipoxygenase baculoviruses were used to infect spodoptera frugiperda insect cells (sf9). at 54 h post-infection, maximal 12-lipoxygenase activity and protein levels were achieved; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in w ... | 1993 | 8319693 |
| nucleotide sequence of the ubiquitin-39k gene region from the orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome. | the region of the orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (opmnpv) genome containing the ubiquitin and the nuclear matrix-associated protein (39k) genes was sequenced. the first 77 amino acids of the opmnpv ubiquitin open reading frame (orf) showed 84.4% and 79% amino acid sequence identity to autographa californica multinucleocapsid nuclear polyhedrosis virus (acmnpv) and the spodoptera frugiperda insect ubiquitin, respectively. the predicted opmnpv ubiquitin protein c ... | 1993 | 8389803 |
| recovery and characterization of a rotavirus outer capsid protein expressed in a recombinant insect cell system. | recombinant osu vp4 protein, and outer capsid antigen of porcine rotavirus, was purified to a high level from the spent broth of baculovirus-infected spodoptera frugiperda insect cells. initial clarification of the broth with a 0-60% ammonium sulfate cut retained 93% of the total vp4. q-sepharose ion exchange chromatography performed at ph 6.5 yielded 67% of the initial amount of vp4 in the pooled fractions, with more than four times the purity of the original sample. gel filtration chromatograp ... | 1993 | 8390880 |
| expression of the bovine viral diarrhoea virus osloss p80 protein: its use as elisa antigen for cattle serum antibody detection. | the putative gene encoding the cytopathic bovine viral diarrhoea virus (bvdv) osloss strain p80 protein was amplified by pcr and inserted into a t7 promoter-based vector for expression in escherichia coli. bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. rabbit antisera were raised against this p80 recombinant antigen and assayed for the immunoprecipitation of either p120 or p80 protein from cytopathic or non-cyt ... | 1993 | 8393083 |
| synthesis of immunogenic hepatitis a virus particles by recombinant baculoviruses. | recombinant baculoviruses were constructed which contained the hepatitis a virus (hav) open reading frame (orf) under the control of the polyhedrin promoter. northern blot analysis with an hav-specific oligonucleotide probe demonstrated a single transcript large enough to include the hav orf in spodoptera frugiperda cells infected with these recombinants. immunoblots revealed a 220 kda protein representing the hav polyprotein. in addition, proteins which co-migrated with hav capsid proteins, and ... | 1993 | 8393604 |
| the multifunctional folic acid synthesis fas gene of pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase. | the nucleotide sequence of a folic acid synthesis (fas) gene from pneumocystis carinii contains an open reading frame (orf) that predicts a protein of 740 amino acids with an m(r) of 83,979. a recombinant baculovirus was constructed which directed expression of the predicted fas740 polypeptide in cultured spodoptera frugiperda (sf9) insect cells. the overexpressed 'full-length' protein migrated anomalously in sodium dodecyl sulfate/polyacrylamide gels, with an apparent molecular mass of 71.5 kda ... | 1993 | 8397083 |
| isolation, characterization, and proteolysis of human prosaposin, the precursor of saposins (sphingolipid activator proteins). | prosaposin contains separate domains in tandem for four saposins, a, b, c, and d. these mature saposins are produced by limited proteolysis of prosaposin. they are involved in lysosomal hydrolysis of gm1 ganglioside, gluco- and galactocerebrosides, sulfatides, and sphingomyelin and other sphingolipids. prosaposin also exists as a secretory protein in body fluids. in this investigation prosaposin was expressed in spodoptera frugiperda cells (sf9) by infection with baculovirus containing a full le ... | 1993 | 8323276 |
| expression of feline immunodeficiency virus gag and env precursor proteins in spodoptera frugiperda cells and their use in immunodiagnosis. | the gag and env genes of the feline immunodeficiency virus strain ut113 were cloned into a baculovirus transfer vector. the recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (sf9 cells). leader sequence cleavage occurred in sf9 cells expressing the envelope precursor, but further processing was not observed. crude lysates of insect cells infected with the wild-type baculovirus or with the recombinant viru ... | 1993 | 8408554 |
| expression of anticardiolipin cofactor, human beta 2-glycoprotein i, by a recombinant baculovirus/insect cell system. | a full-length cdna coding a human beta 2-glycoprotein i (beta 2-gpi) was introduced into the baculovirus genome to construct a recombinant baculovirus. spodoptera frugiperda (sf9) cells were infected with the recombinant baculovirus. a protein (mol. wt 43,000) reactive with anti-beta 2-gpi antisera was produced in the insect cells and secreted into the culture medium. the recombinant beta 2-gpi was purified from the culture supernatant by sequential cardiolipin (cl)-affinity column chromatograph ... | 1993 | 8324900 |
| immunocytochemical characterization of p24, a baculovirus capsid-associated protein. | an open reading frame (orf 1) located upstream of the polyhedron envelope protein gene in the orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (opmnpv) genome was cloned in-frame into a trpe bacterial expression vector. the fusion protein produced by this construct was used for the preparation of a monospecific antiserum. western blot analysis of extracts from opmnpv-infected lymantria dispar cells and autographa californica npv (acmnpv)-infected spodoptera frugiperda cells dete ... | 1993 | 8423444 |
| antibody response to the m2 protein of influenza a virus expressed in insect cells. | a recombinant baculovirus expressing the m2 protein from influenza a/ann arbor/6/60 (h2n2) virus (aa60 virus) was constructed. the expressed m2 protein was recognized by a monoclonal antibody specific for the m2 protein and comigrated with the m2 protein from cells infected with aa60 virus on sds-polyacrylamide gels. immunofluorescence studies indicated that the expressed m2 protein was present on the surface of spodoptera frugiperda (sf9) cells infected with the recombinant baculovirus. immunoa ... | 1993 | 8423445 |
| immunoreactivity and protective effects in mice of a recombinant dengue 2 tonga virus ns1 protein produced in a baculovirus expression system. | the double-stranded replicative form of dengue 2 virus (den-2) rna (tonga strain) was used as a substrate to produce dna clones of the ns1-ns2a genes via reverse transcriptase synthesis of full length cdna followed by polymerase chain reaction amplification of the ns1-ns2a region. products were cloned into ptz18r for sequencing and into baculovirus for expression studies. the deduced amino acid sequence of the ns1-ns2a was almost identical to that of the s1 attenuated strain of den-2 puerto rico ... | 1993 | 8423452 |
| phosphorylation of elongation factor-2 from the lepidopteran insect, spodoptera frugiperda. | in mammalian cells, protein synthesis can be regulated at the level of elongation by the phosphorylation of elongation factor 2 (eef-2) by a highly specific ca2+/calmodulin-dependent kinase. in this report, we show that eef-2 from a cell line derived from the insect, spodoptera frugiperda, is a substrate for mammalian eef-2 kinase and that phosphorylation is ca(2+)-dependent. furthermore, two-dimensional peptide mapping shows that the kinase phosphorylates the same sites in spodoptera eef-2 as t ... | 1993 | 8335099 |
| one-step purification and characterization of human pancreatic lipase expressed in insect cells. | a cdna clone encoding the sequence of human pancreatic lipase (hpl) was subcloned into the baculovirus transfer vector pvl1392 and used in co-transfection of spodoptera frugiperda (sf9) insect cells with wild-type autographa californica nuclear polyhedrosis virus (acnpv) dna. a single recombinant protein (50 kda) secreted by sf9 cells was detectable in the culture medium 24 h post-infection using both anti-hpl polyclonal antibodies and potentiometric measurements of lipolytic activity. the expre ... | 1993 | 8335100 |
| immediate-early baculovirus genes transactivate the p143 gene promoter of autographa californica nuclear polyhedrosis virus. | trans-acting regulatory components of autographa californica nuclear polyhedrosis virus (acmnpv) were studied in a transient assay system for their ability to activate the p143 gene promoter. a region including 502 nt upstream of the aug of the p143 gene was linked to the bacterial chloramphenicol acetyltransferase (cat) gene. the p143 promoter-cat construct was used to identify acmnpv immediate-early gene products required for expression from the p143 gene early promoter. transient expression a ... | 1993 | 8337840 |
| overexpression, characterization, and purification of a recombinant mouse immunophilin fkbp-52 and identification of an associated phosphoprotein. | to gain insight into the structure and function of the immunophilin fkbp-52, a mouse fkbp-52 was overexpressed in spodoptera frugiperda insect cells (sf9 cells) with the baculovirus expression system. the purification and characterization of the recombinant fkbp-52 (rfkbp-52) was facilitated by incorporating a histidine 6-mer domain at its n terminus. the rfkbp-52 was highly purified on a n(i)2+ affinity resin with an estimated recovery of 10 mg of pure protein from 1 liter of sf9 cell culture. ... | 1993 | 8341706 |
| cdna cloning and expression of a soluble epoxide hydrolase from human liver. | we report the cloning and expression of a cdna that encodes a soluble epoxide hydrolase from human liver. the 2101-base clone predicts a 554-residue protein (m(r) 62,640) with an apparently imperfect peroxisomal targeting signal of ser-lys-met at the carboxy terminus. the cdna was expressed in the baculovirus system in the spodoptera frugiperda 21 cell line. the recombinant protein was similar to soluble epoxide hydrolase isolated from human liver in terms of molecular weight, hydrolytic activit ... | 1993 | 8342951 |
| 5-lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-lipoxygenase. | 5-lipoxygenase (5-lo) and its activating protein (flap) are both required for cellular leukotriene (lt) synthesis, with 5-lo catalyzing both the synthesis of (5s)-5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-hpete) from arachidonic acid and the subsequent synthesis of lta4 from 5-hpete. we have previously expressed both human 5-lo and human flap to high levels in spodoptera frugiperda (sf9) insect cells, using recombinant baculoviruses. to study the mechanism by which flap activates 5-lo, we ... | 1993 | 8344271 |
| bovine respiratory syncytial virus nucleocapsid protein expressed in insect cells specifically interacts with the phosphoprotein and the m2 protein. | the coding region of the gene for the nucleocapsid (n) protein of bovine respiratory syncytial virus has been inserted into the genome of autographa californica nuclear polyhedrosis virus (acnpv) using transfer vector pvl 1393. infection of spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of n protein. this protein was indistinguishable from the authentic bovine respiratory syncytial virus (brsv) n protein by sds-gel electrophoresis and immunoprecipitat ... | 1993 | 8438581 |
| biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected sf21 cells. characterization of polypeptides and posttranslational modifications. | human interleukin 2 (il-2) and human il-2 mutant proteins, with artificially introduced n-glycosylation or o-glycosylation sites, have been expressed in a lepidopteran cell line (sf21, spodoptera frugiperda) using recombinant baculovirus vectors. only approximately 25% of the total recombinant il-2 protein synthesized by sf21 cells was secreted into the culture medium. significant n-terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules). alanine and proline ... | 1993 | 8344280 |
| expression and characterization of biologically active human hepatocyte growth factor (hgf) by insect cells infected with hgf-recombinant baculovirus. | a cdna containing the entire coding sequence of human hepatocyte growth factor (hgf) [also known as scatter factor (sf)] was inserted into the genome of autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. insect cells (spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/l) of biologically active hgf into the culture medium. the recombinant hgf induces pronounced morphological ... | 1993 | 8347597 |
| the presence of udp-n-acetylglucosamine:alpha-3-d-mannoside beta 1,2-n-acetylglucosaminyltransferase i activity in spodoptera frugiperda cells (iplb-sf-21ae) and its enhancement as a result of baculovirus infection. | a golgi preparation from spodoptera frugiperda (iplb-sf-21ae) cells was incubated in the presence of the mannosidase ii inhibitor, swainsonine, with the oligosaccharide, m(alpha 1,3)[[m(alpha 1,3)[m(alpha 1,6)]m(alpha 1,6)]] m(beta 1,4)gn(beta 1,4)gn (m5gn2), the preferred substrate for the enzyme, udp-n-acetylglucosamine:alpha-3-d-mannoside beta 1,2-n-acetylglucosaminyltransferase i (gn-ti). this resulted in formation of the product, gn(beta 1,2)m(alpha 1,3)[[m(alpha 1,3)[m(alpha 1,6)]m(alpha 1 ... | 1993 | 8349674 |
| inhibition of african swine fever virus binding and infectivity by purified recombinant virus attachment protein p12. | the african swine fever virus protein p12, involved in virus attachment to the host cell, has an apparent molecular mass of 17 kda in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. we have also identified 12- and 10-kda forms of the p12 protein in infected vero cells and found that the mature 17-kda protein is the only form present in virus particles. the p12 protein has been produced in large amounts in spodoptera frugiperda insect cells infected with a ... | 1993 | 8350406 |
| expression of human il-2 receptor alpha- and beta-chains using the baculovirus expression system. | the genes encoding the alpha- and beta-chains of the human interleukin-2 receptor were expressed in lepidopteran insect cells using the baculovirus expression vector system. the corresponding genes were inserted under the polyhedrin promoter of the autographa california nuclear polyhedrosis virus and expressed in the spodoptera frugiperda insect cell line during viral infection. the recombinant receptor proteins were identified in the insect cell lysates by using protein dot blot and elisa techn ... | 1993 | 8356402 |
| steroid requirement for androgen receptor dimerization and dna binding. modulation by intramolecular interactions between the nh2-terminal and steroid-binding domains. | infection of spodoptera frugiperda sf9 insect cells with recombinant human androgen receptor (ar) baculovirus results in expression of a 118-kda phosphoprotein that displays high affinity androgen binding and androgen-dependent targeting to the nucleus. using the dna mobility shift assay, specific in vitro binding of full-length ar to androgen response element dna (are) requires intracellular hormone exposure. the ability of a variety of steroids to induce are binding paralleled their transcript ... | 1993 | 8360187 |
| baculovirus-mediated expression of human 65 kda and 67 kda glutamic acid decarboxylases in sf9 insect cells and their relevance in diagnosis of insulin-dependent diabetes mellitus. | cdnas coding for the full-length human 65 and 67 kda glutamic acid decarboxylases (gad65 and gad67) were amplified from pancreas and hippocampus cdna libraries by polymerase chain reaction, respectively. both cdnas were inserted into a baculovirus vector which mediated highly efficient expression of the human gad65 and gad67 with histidine-hexapeptides as affinity ligands at their c-termini in spodoptera frugiperda (sf9) cells. the recombinant gad proteins were purified to homogeneity by affinit ... | 1993 | 8370667 |
| functional expression of the rat anion exchanger ae2 in insect cells by a recombinant baculovirus. | we used baculovirus to transiently express a rat anion exchanger (ae2) in spodoptera frugiperda (sf9) insect cells. no detectable cl(-)-hco3- exchange activity was observed in wild type or sham-infected sf9 cells, monitored using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, a ph-sensitive fluorescent dye. functional expression of anion exchange activity in the ae2 recombinant baculovirus-infected cells was observed within the first day after infection and sustained over the next 3 days. th ... | 1993 | 8476013 |
| sequence and temporal appearance of the early transcribed baculovirus gene he65. | we have identified the early transcribed he65 gene by screening a cdna library from polyadenylated rna which was isolated at 1 h after infection of spodoptera frugiperda cells with autographa californica nuclear polyhedrosis virus (acnpv). nucleotide sequencing analysis of the he65-specific cdna clone reveals one open reading frame of 1,662 nucleotides from which a protein of 65 kda in size can be predicted. the he65 gene is located downstream of the late transcribed p80 gene and upstream of the ... | 1993 | 8371346 |
| adenine dna methyltransferase m.cviri expression accelerates apoptosis in baculovirus-infected insect cells. | the adenine dna methyltransferase m.cviri (tgcma) gene from chlorella virus xz-6e was cloned into the autographa californica nuclear polyhedrosis virus (acmnpv) genome and expressed in spodoptera frugiperda insect cells under the control of two tandemly arranged viral promoters, the early etl promoter and the late polyhedrin promoter. m.cviri activity was first detected at 10 hr p.i and reached a maximum at 48 hr p.i. viral dna synthesized in insect cells infected with m.cviri expressing virus ( ... | 1993 | 8372450 |
| identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression. | an autographa californica nuclear polyhedrosis virus (acmnpv) gene required in transient expression assays for late and very late viral gene expression was identified, sequenced, and transcriptionally mapped. this gene, designated late expression factor 1 (lef-1), was located between 7.4 and 8.7 map units of the acmnpv physical map. it was identified by cotransfecting spodoptera frugiperda cultured cells with a collection of overlapping cloned dna fragments covering the entire acmnpv genome and ... | 1993 | 8497062 |
| formation of poliovirus-like particles by recombinant baculoviruses expressing the individual vp0, vp3, and vp1 proteins by comparison to particles derived from the expressed poliovirus polyprotein. | the cdna sequences encoding the vp0, vp3, and vp1 structural proteins of poliovirus type 3 (strain p3/leon/37) have been individually cloned and used to prepare recombinant baculoviruses based on autographa californica nuclear polyhedrosis viruses (acnpv). expression of the proteins was monitored in virus-infected spodoptera frugiperda cells. both the individually expressed vp0 protein and vp0 derived from the complete poliovirus coding region were shown to incorporate myristic acid. a significa ... | 1993 | 8380663 |
| isolation and expression of human 1,25-dihydroxyvitamin d3 24-hydroxylase cdna. | human 1,25-dihydroxyvitamin d3 24-hydroxylase cdna clones were isolated from an hl-60 cell cdna library by using a reverse transcription/polymerase chain reaction-generated human cdna probe. the 24-hydroxylase cdna consists of a 1539-bp open reading frame encoding a 513-amino acid polypeptide. protein sequence analysis shows that the human 24-hydroxylase is 90% homologous (82% identical) to that of the rat, with 100% homology in the 21-amino acid heme-binding region. northern blot analysis showe ... | 1993 | 8506296 |
| breaching the conformational integrity of the catalytic triad of the serine protease plasmin: localized disruption of a side chain of his-603 strongly inhibits the amidolytic activity of human plasmin. | site-directed mutagenesis has been used to construct a cdna that encodes a recombinant variant human plasminogen (hpg) containing a pro-611-->ile mutation (mrhpg). the mutein was expressed in recombinant baculovirus-infected spodoptera frugiperda cells (iplb-sf-21ae), and purified. after activation of this zymogen to its corresponding form of the serine protease plasmin (mrhpm), this latter enzyme was essentially inactive toward an amide plasmin substrate, most likely from alteration of the spat ... | 1993 | 8506386 |
| expression of rat ctp:phosphocholine cytidylyltransferase in insect cells using a baculovirus vector. | ctp:phosphocholine cytidylyltransferase (ct) is a key regulatory enzyme in phosphatidylcholine biosynthesis. we constructed a recombinant baculovirus (bct) containing rat ct cdna under the control of the polyhedrin promoter. crude cell extracts of spodoptera frugiperda (sf9) cells infected with bct possessed 250-fold higher specific activities for ct compared to rat liver cytosol, and ct protein constituted 3-6% of the total cellular protein. the 42-kda form of ct predicted from the cdna sequenc ... | 1993 | 8382903 |
| functional expression of the alpha 2 and alpha 3 isoforms of the na,k-atpase in baculovirus-infected insect cells. | multiple isoforms of both the alpha and beta subunits of na,k-atpase have been identified. elucidating their roles has been complicated by the fact that most tissues express multiple isoforms and purification techniques specific for each isoform have not been achieved. the baculovirus expression system, which uses the baculovirus autographica californica to infect insect cells, is an ideal system for studying the na,k-atpase isoforms since high amounts of foreign proteins can be produced and som ... | 1993 | 8383329 |
| interaction of hepatic microsomal epoxide hydrolase derived from a recombinant baculovirus expression system with an azarene oxide and an aziridine substrate analogue. | a recombinant baculovirus (vehx) encoding rat hepatic microsomal epoxide hydrolase has been constructed. infection of spodoptera frugiperda (sf9) cells with the recombinant virus results in the expression of the enzyme at a level estimated to be between 5% and 10% of the cellular protein. the enzyme, which can be purified in 15% yield by a simple three-step procedure involving detergent extraction, deae-cellulose chromatography, and removal of the detergent on hydroxylapatite, has physical and k ... | 1993 | 8383521 |
| location of two putative origins of dna replication of autographa californica nuclear polyhedrosis virus. | previously, we described a defective autographa californica nuclear polyhedrosis virus (acmnpv) which must contain cis-acting elements required for dna synthesis, such as the origin(s) of replication (ori). defective genomes of acmnpv generated after serial undiluted passage were analyzed further. three small separated regions were retained in dna of defective acmnpv and accumulated in extracellular defective interfering viruses as well as in intracellular dna after 40 passages. two of these reg ... | 1993 | 8517035 |
| expression and characterization of the rat d3 dopamine receptor: pharmacologic properties and development of antibodies. | a baculovirus expression system provided an enriched source of biologically and immunologically active d3 dopamine receptors. receptors expressed in spodoptera frugiperda insect (sf9) cells at a density of 5 to 15 pmol/mg of protein displayed high affinity for the antagonists, eticlopride, fluphenazine and spiroperidol, and the agonist, n-propylnorapomorphine. the binding of agonists was not sensitive to gtp. antisera raised against synthetic peptides in the third intracellular loop of the d3 do ... | 1993 | 8437101 |
| 5-lipoxygenase-activating protein is an arachidonate binding protein. | 5-lipoxygenase-activating protein (flap) is an 18-kda integral membrane protein which is essential for cellular leukotriene (lt) synthesis, and is the target of lt biosynthesis inhibitors. however, the mechanism by which flap activates 5-lo has not been determined. we have expressed high levels of human flap in spodoptera frugiperda (sf9) insect cells infected with recombinant baculovirus, and used this system to demonstrate that flap specifically binds [125i]l-739,059, a novel photoaffinity ana ... | 1993 | 8440384 |
| functional human transcobalamin ii isoproteins are secreted by insect cells using the baculovirus expression system. | transcobalamin ii (tcii) is a cobalamin (cbl, vitamin b12)-binding protein in mammalian plasma that facilitates the cellular uptake of the vitamin. to obtain human tcii in sufficient quantity for analytical studies, the complementary dna (cdna) encoding tcii was inserted into the plasmid pvl 1393, and the baculovirus expressing tcii was obtained by homologous recombination in spodoptera frugiperda (sf9) insect cells by cotransfection with the wildtype virus. under optimized conditions, sf9 cells ... | 1993 | 8443384 |
| the protoxin composition of bacillus thuringiensis insecticidal inclusions affects solubility and toxicity. | most bacillus thuringiensis strains producing toxins active on lepidoptera contain several plasmid-encoded delta-endotoxin genes and package related protoxins into a single inclusion. it was previously found that in b. thuringiensis subsp. aizawai hd133, which produces an inclusion comprising the cryiab, cryic, and cryid protoxins, there is a spontaneous loss in about 1% of the cells of a 45-mda plasmid containing the cryiab gene. as a result, inclusions produced by the cured strain were less re ... | 1995 | 8526519 |
| an apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. | spodoptera frugiperda sf-21 cells infected with autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. to identify p35-homologous genes in other baculoviruses, a. californica nuclear polyhedrosis virus dna containing a deletion in p35 was cotransfected into sf-21 cells along with genomic dnas from other baculoviruses. one of the viral dnas which were able to rescue wild-type infection was from cydia pomonella ... | 1993 | 8445726 |
| purification and renaturation of japanese encephalitis virus nonstructural glycoprotein ns1 overproduced by insect cells. | the nonstructural protein ns1 of japanese encephalitis virus is a major immunogen produced during flavivirus infection. however, the function of this protein has not been identified. to analyze its biochemical properties and evaluate its potential activity in the virus life cycle, the protein was produced in spodoptera frugiperda insect cells (sf9), using a recombinant baculovirus, and purified. as described previously by m. flamand, v. deubel, and m. girard (1992, virology 191, 826-836), a smal ... | 1995 | 8527939 |
| convenient desktop-scale production of the extracellular domain of the human growth hormone receptor by an insect-baculovirus secretion system using a protein-free culture. | expression of a gene encoding the extracellular domain of the human growth hormone receptor (hghr-ed) inserted into the genome of autographa californica nuclear polyhedrosis virus was done using a desktop-scale spinner culture. spodoptera frugiperda 9 (sf9) cells infected with the recombinant virus secreted a protein with hgh-binding activity into the medium. oxygen supplementation was required for high level secretion of the product. the highest cell production capability was estimated at more ... | 1995 | 8534995 |
| expression of a 32 kilodalton theileria sergenti piroplasm surface protein by recombinant baculoviruses. | previous studies detected a single amino acid substitution (ala196 to gly196) between cdna clones encoding a 32 kda antigen (p32) of theileria sergenti (chitose stock) obtained from a persistently infected calf. in this study, 2 different recombinant baculoviruses (pac/p32-ala196 and pac/p32-gly196) were constructed for the expression of p32. molecular masses of the polypeptides produced in spodoptera frugiperda cells infected with the recombinant baculoviruses were the same as that of authentic ... | 1995 | 8550294 |
| characterization of vaccinia surface antigen expressed by recombinant baculovirus. | the gene encoding the vaccinia surface antigen (s antigen) was inserted into a baculovirus transfer vector and a recombinant virus was isolated. the s antigen was expressed on the surface of spodoptera frugiperda cells (sf cells) infected with the recombinant baculovirus. recombinant proteins were detected in immunoblotting with anti-vaccinia serum and have the apparent molecular weight of 40 and 50-kda. the 50-kda polypeptide was tunicamycin sensitive and was thus glycosylated. the glycosylated ... | 1993 | 8460484 |
| the expression of hemolytically active human complement protein c9 in mammalian, insect, and yeast cells. | the cdna sequence encoding mature human c9 protein and its signal peptide was cloned into three expression vectors for expression in cos-7 (mammalian), spodoptera frugiperda iplb-sf-21ae (insect), and saccharomyces cerevisiae (yeast) cells. in addition, c9 cdna encoding only the mature protein was fused to the yeast invertase leader sequence (suc2) and cloned for expression in yeast. under optimal conditions cos-7 and iplb-sf-21ae cells secreted recombinant c9 (rc9) at concentrations of about 11 ... | 1993 | 8471847 |
| use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus. | flock house virus (fhv) is a small icosahedral insect virus of the family nodaviridae. its genome consists of two messenger-sense rna molecules, both of which are encapsidated in the same particle. rna1 (3.1 kb) encodes proteins required for viral rna replication; rna2 (1.4 kb) encodes protein alpha (43 kda), the precursor of the coat protein. when spodoptera frugiperda cells were infected with a recombinant baculovirus containing a cdna copy of rna2, coat protein alpha assembled into viruslike ... | 1993 | 8474173 |
| ionic permeabilities induced by bacillus thuringiensis in sf9 cells. | the effect of bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular ph (phi) of individual sf9 cells of the lepidopteran species spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (pbfi) and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (bcecf). the sequential removal of k+ and na+ from the medium, in the presence of cryic, a toxin which is highly active against sf9 cells, caused s ... | 1995 | 8558602 |
| isolation of the full-length murine erythropoietin receptor using a baculovirus expression system. | the full-length murine erythropoietin receptor was expressed in spodoptera frugiperda (sf9) cells using a recombinant baculovirus vector. erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kd. erythropoietin receptors produced in sf9 cells could be solubilized using chaps in a form capable of binding erythropoietin, and the solubilized receptor bound to im ... | 1996 | 8562964 |
| intrinsic glycosylation potentials of insect cell cultures and insect larvae. | the glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared. the spodoptera frugiperda (sf21) and trichoplusia ni (tn-368 and bti-tn-5b1-4) cell lines possessed several intrinsic glycoproteins that are modified with both n- and o-linked oligosaccharides. the n-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types. similarly, the t. ni l ... | 1995 | 8564076 |
| baculovirus expression of the maize mitochondrial protein urf13 confers insecticidal activity in cell cultures and larvae. | the urf13 protein, which is encoded by the mitochondrial gene t-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the texas male-sterile cytoplasm (cms-t) of maize. mitochondrial sensitivity to two host-specific fungal toxins (t toxins) is mediated by the interaction of urf13 and t toxins to form pores in the inner mitochondrial membrane. a carbamate insecticide, methomyl, mimics the effects of t toxins on isolated cms-t mitochondria. urf13 was expressed in spodo ... | 1993 | 8475086 |
| the 35-kilodalton protein gene (p35) of autographa californica nuclear polyhedrosis virus and the neomycin resistance gene provide dominant selection of recombinant baculoviruses. | autographa californica nuclear polyhedrosis virus (acmnpv) recombinants were constructed to test the effectiveness of the acmnpv 35-kilodalton protein gene (35k gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. insertion of the acmnpv apoptosis suppressor gene (p35) into the genome of p35-deletion mutants inhibited premature host cell death and increased virus yields up to 1200-fold at low multiplicities in spodoptera frugiperda (sf21) cell ... | 1993 | 8493093 |
| preparation of monoclonal antibodies against marek's disease virus serotype 1 glycoprotein d expressed by a recombinant baculovirus. | a recombinant baculovirus, the genome of which contains dna encoding marek's disease virus serotype 1 (mdv1) homolog of glycoprotein d (gd) of herpes simplex virus under the polyhedrin promoter was constructed and designated racmdv1gd. five monoclonal antibodies (mabs) which recognize the mdv1 homolog of gd (mdv1 gd) in spodoptera frugiperda cells infected with racmdv1gd were prepared. the mabs reacted with proteins ranging from 52 to 49 kda in racmdv1gd-infected cell lysates by immunoblot analy ... | 1995 | 8578860 |
| expression of rat liver phenylalanine hydroxylase in insect cells and site-directed mutagenesis of putative non-heme iron-binding sites. | rat liver phenylalanine hydroxylase was expressed in both escherichia coli and the spodoptera frugiperda insect cell line, sf9. recombinant enzyme from e. coli was inactive and contained less than 0.1 iron atom/subunit. in contrast, recombinant enzyme expressed in sf9 cells using a baculovirus vector was active and identical in several properties to phenylalanine hydroxylase from rat liver: the km for 6-methyltetrahydropterin was 39 microm (compared with 35 microm for the rat liver enzyme), 1 at ... | 1993 | 8385134 |
| human beta 2-adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous gtp-binding protein to adenylyl cyclase. | spodoptera frugiperda insect cells (sf9) containing the stably integrated human beta 2-adrenergic receptor gene under the control of the baculovirus ie1 promoter expressed up to 350,000 human receptors/cell. the number of receptors did not change with cell density or age of culture. the adrenergic receptors overexpressed in the insect cells were functional with respect to their ligand binding and signalling properties. coupling of the receptors to endogenous gtp-binding proteins is demonstrated ... | 1993 | 8386637 |
| purification of posttranslationally modified and unmodified rab5 protein expressed in spodoptera frugiperda cells. | | 1995 | 8583943 |
| possible role of phosphorylation in the function of chicken myod1. | chicken myod1 (cmd1), an equivalent to the mouse myod1, expressed in chicken skeletal muscle (lin, z.-y., dechesne, c. a., eldridge, j., and paterson, b. m. (1989) genes & dev. 3, 986-996), was produced in spodoptera frugiperda (sf9) cells by baculovirus expression vector and purified to almost homogeneity. this cmd1 was directly demonstrated to be a phosphoprotein by a 32p-labeling experiment. phosphoamino acid analysis revealed that only serine residue was phosphorylated. phosphoamino acid of ... | 1993 | 8389359 |
| purification of the bovine nicotinic acetylcholine receptor alpha-subunit expressed in baculovirus-infected insect cells. | the bovine skeletal muscle nicotinic acetylcholine receptor alpha-subunit was produced in insect, spodoptera frugiperda (sf9), cells by infection with a recombinant baculovirus. the expressed alpha-subunit protein could not be solubilized efficiently with triton x-100 or sodium cholate, but could be solubilized efficiently with zwittergent 3-14, sodium dodecyl sulfate or tris(hydroxymethyl)aminomethane dodecylsulfate. after solubilization of the alpha-subunit protein with zwittergent 3-14 from t ... | 1993 | 8407866 |
| mosquitocidal activity of the cryic delta-endotoxin from bacillus thuringiensis subsp. aizawai. | the cloned 135-kda cryic delta-endotoxin from bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against spodoptera litoralis and spodoptera frugiperda larvae and in vitro against the s. frugiperda sf9 cell line. here, we report that the cryic delta-endotoxin cloned from b. thuringienesis subsp. aizawai hd-229 and expressed in an acrystalliferous b. thuringiensis strain is also toxic to aedes aegypti, anophles gambiae, and culex quinquefasciatus mosquito larv ... | 1996 | 8593070 |
| post-translational processing of barley beta-glucan endohydrolases in the baculovirus-insect cell expression system. | two cdnas encoding barley (1-->3,1-->4)-beta-glucanase (ec 3.2.1.73) isoenzymes ei and eii have been expressed in spodoptera frugiperda (sf9) cell cultures using the baculovirus acnpv vector. modifications to both the 5' and 3' ends of the cdnas were required before satisfactory levels of expression were obtained. the modified cdnas directed high levels of (1-->3,1-->4)-beta-glucanase expression in the sf9 insect cell cultures, with yields of approximately 10 mg/liter of isoenzyme ei (expei) and ... | 1993 | 8422276 |
| expression of bombyx mori cytoplasmic polyhedrosis virus polyhedrin in insect cells by using a baculovirus expression vector, and its assembly into polyhedra. | a cdna encoding the cytoplasmic polyhedrin of bombyx mori cytoplasmic polyhedrosis virus (bmcpv) strain h was introduced into an improved baculovirus expression vector which can be utilized to express foreign genes in the spodoptera frugiperda cell line iplb-sf-21ae (sf21 cells) and the b. mori cell line bmn. a recombinant virus produced large, cubic inclusion body-like structures in infected sf21 and bmn cells. western blot analysis showed that these structures were bmcpv polyhedra. this result ... | 1993 | 8423453 |
| expression of biologically active human factor viii using a baculovirus vector. | factor viii is a complex, plasma glycoprotein involved in the process of blood coagulation. production of the recombinant molecule has largely been confined to mammalian cell systems which have, in general, proven to be inefficient producers of factor viii. the use of a baculovirus expression system may provide increased levels of this glycoprotein, although it is not certain that insect cell-derived factor viii will be biologically active. the n-linked glycosylation patterns in insect cells, un ... | 1993 | 8427596 |
| production of recombinant androgen receptor in a heterologous expression system. | to facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in spodoptera frugiperda (sf9) insect cells, using the baculovirus expression system. recombinant baculovirus dna-infected sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. only < 1% of the recombinant protein was soluble in low-salt buffers; the majority formed ele ... | 1993 | 8432026 |
| site-directed mutagenesis and chemical modification of histidine residues on an alpha-class chick liver glutathione s-transferase cl 3-3. histidines are not needed for the activity of the enzyme and diethylpyrocarbonate modifies both histidine and lysine residues. | each chick liver glutathione s-transferase cl 3 subunit contains three histidine residues: his142, his158 and his228. cl 3-3 can be inactivated by treating with diethylpyrocarbonate. the inactivation process is ph dependent and the pka of the modified residue is 6.4. the second-order inhibition rate constant is 741 m-1min-1 at ph 7.0. based on difference-spectrum and kinetic analysis, inactivation coincides with the modification of one histidine residue. however, hydroxylamine treatment of the d ... | 1993 | 8436137 |
| expression of human thromboxane synthase using a baculovirus system. | human thromboxane (tx) synthase (ec 5.3.99.5) was produced by the baculovirus expression system using cdna encoding human tx synthase [(1991) biochem. biophys. res. commun. 78, 1479-1484]. a recombinant baculovirus txs7 was expressed in spodoptera frugiperda sf9 insect cells. the expressed protein was recognized by monoclonal antibody, kon 7 raised against human tx synthase [(1990) blood 76, 80-85]. the recombinant tx synthase catalyzed the conversion of prostaglandin (pg) h2 to txa2 and 12-hydr ... | 1993 | 8436233 |
| high level expression of the large subunit of mouse ribonucleotide reductase in a baculovirus system. | the large subunit of ribonucleotide reductase from mouse has been overexpressed in spodoptera frugiperda cells infected with recombinant baculovirus. the expressed protein was purified by affinity chromatography to apparent homogeneity as determined by sds-page. the homogeneous protein is recognized in western blot analysis by a monoclonal antibody raised to the large subunit of ribonucleotide reductase from calf thymus, has the correct n-terminal sequence, and, in the presence of the small subu ... | 1993 | 8495754 |
| post-translational processing and thr-206 are required for glycosylasparaginase activity. | lysosomal glycosylasparaginase is encoded as a 36.5 kda polypeptide that is post-translationally processed to subunits of 19.5 kda (heavy) and 15 kda (light). recombinant glycosylasparaginase has been expressed in spodoptera frugiperda insect cells enabling the precursor and processed forms to be isolated and their catalytic potential determined. only the subunit conformation was functional indicating glycosylasparaginase is encoded as an inactive zymogen. the newly created amino terminal residu ... | 1993 | 8500622 |
| apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus. | apoptotic programmed cell death occurs when the insect cell line sf-21, derived from spodoptera frugiperda, is infected with mutants of the baculovirus autographa californica nuclear polyhedrosis virus (acmnpv) which lack a functional p35 gene. however, infection of the trichoplusia ni tn-368 cell line with p35 mutants does not result in apoptosis (r. clem, m. fechheimer, and l. miller, science 254:1388-1390, 1991). we have examined the effect of apoptosis on acmnpv infections in cell lines and ... | 1993 | 8510202 |
| in vitro transcription from baculovirus late gene promoters: accurate mrna initiation by nuclear extracts prepared from infected spodoptera frugiperda cells. | extracts prepared from nuclei of autographa californica nuclear polyhedrosis virus-infected spodoptera frugiperda cells were shown to support in vitro transcription from baculovirus late gene promoters. in vitro transcription was optimized for the late promoter of the 39k gene. the mg2+ concentration was critical; concentrations higher than 1 to 2 mm did not support late transcription. additional conditions included template (40 micrograms/ml), extract (2.5 mg/ml), and incubation time (25 min). ... | 1993 | 8510206 |
| high-level production of biologically active human cytosolic phospholipase a2 in baculovirus-infected insect cells. | infection of spodoptera frugiperda insect cells with a recombinant baculovirus expressing human cytosolic phospholipase a2 (cpla2) resulted in the production of biologically active protein. the level of recombinant human cpla2 production in infected insect cells was at least 50-fold higher than that observed in human monoblast u937 cells. | 1993 | 8514199 |
| purification of recombinant glur-d glutamate receptor produced in sf21 insect cells. | glur-d glutamate receptors carrying flag and polyhis affinity tags at the n-terminus and c-terminus, respectively, were expressed in recombinant baculovirus-infected spodoptera frugiperda sf21 cells. affinity-tagged receptors displayed ligand-binding affinity (kd = 40 nm) and an expression level (bmax 10-30 pmol/mg protein) similar to that of insect-cell-expressed wild-type glur-d, as determined by [3h]-alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid ([3h]ampa) binding. the receptor wa ... | 1995 | 8521834 |
| expression of functional ricin b chain using the baculovirus system. | the ricin b chain (rtb) was expressed using a baculovirus expression system. the rtb coding sequence downstream of the preproricin signal sequence was inserted in the baculovirus transfer vector pm34t. after cotransfection of spodoptera frugiperda sf9 cells with linearized baculovirus dna, recombinant viruses were selected, cloned and amplified. upon infection of sf9 cells with these recombinant baculoviruses, rtb production was revealed by immunoblotting. rtb expression using this system was op ... | 1995 | 8521841 |
| real-time measurement of cell permeabilization with low-molecular-weight membranolytic agents. | a new method for studying the action of membranolytic agents by simple measurement of light emitted from cells is described. it is based on the expression of the click beetle (pyrophorus plagiophthalamus) luciferase gene (lucgr) in escherichia coli, bacillus subtilis and spodoptera frugiperda cells in order to make them bioluminescent. the diffusion of the substrate for luciferase enzyme through the cell membranes is very low at physiological ph, and therefore a change in membrane permeability i ... | 1995 | 8522460 |
| characterization of human dna ligase i expressed in a baculovirus-insect cell system. | the baculovirus expression system was used to overexpress human dna ligase i (hlig i). approx. 2 mg of recombinant hlig i was produced per 10(8) spodoptera frugiperda sf9 insect cells infected with the recombinant baculovirus. the optimum time point for the production of biologically active recombinant hlig i was 48 h post-infection. lig i activity was demonstrated by auto-adenylating, polynucleotide joining and dna relaxation assays. the baculovirus system has the advantage over previously desc ... | 1995 | 8526875 |
| the nonstructural nsm protein of tomato spotted wilt virus induces tubular structures in plant and insect cells. | the expression and subcellular location of the 33.6-kda nonstructural protein nsm of tomato spotted wilt virus (tswv) was analyzed in nicotiana rustica plants and protoplasts as a function of time. immunofluorescent studies in protoplasts isolated from tswv-infected n. rustica leaves showed that this protein could first be detected close to the periphery of the cell, near the plasmamembrane, and later in tubular structures emerging from the cell surface. in situ, these tubules appeared specifica ... | 1995 | 8553550 |
| comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells. | glucose and glutamine metabolism in several cultured mammalian cell lines (bhk, cho, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. results were compared with data from two insect cell lines and primary liver cells. flux distribution was measured in a representative mammalian (bhk) and an insect (spodoptera frugiperda) cell line using radioactive ... | 1996 | 8557765 |
| identification of a short domain within the non-structural protein ns2 of epizootic haemorrhagic disease virus that is important for single strand rna-binding activity. | the role that a conserved amino acid motif, found in the non-structural protein ns2 of orbiviruses, plays in the interaction of this protein with single stranded (ss) rna was investigated by mutation analysis of the ns2 of epizootic haemorrhagic disease virus. an ns2 mutant in which this motif (amino acids 75 to 83) was deleted was expressed in spodoptera frugiperda cells by a recombinant baculovirus and found to be unable to bind to poly(u)-sepharose. the deletion mutant also differed from wild ... | 1996 | 8558121 |
| human cathepsin o2, a matrix protein-degrading cysteine protease expressed in osteoclasts. functional expression of human cathepsin o2 in spodoptera frugiperda and characterization of the enzyme. | cathepsin o2, a human cysteine protease predominantly present in osteoclasts, has been functionally expressed in spodoptera frugiperda sf9 cells using the autographa californica nuclear polyhedrosis virus. following in vitro activation at ph 4.0 with pepsin, active enzyme with an apparent molecular weight of 29,000 was obtained. n-terminal sequencing revealed the typical processing site for cysteine proteases of the papain family with a proline in the position adjacent to the n-terminal alanine ... | 1996 | 8567669 |
| two human alpha 2-adrenoceptor subtypes alpha 2a-c10 and alpha 2b-c2 expressed in sf9 cells couple to transduction pathway resulting in opposite effects on camp production. | the baculovirus expression vector system utilizing the strong polyhedrin gene promoter of the autographa californica nuclear polyhedrosis virus (acnpv) was used for high level expression of the two alpha 2-adrenoceptor subtypes alpha 2a-c10 and alpha 2b-c2 in spodoptera frugiperda (sf-9) insect cells. for rapid screening of recombinant viruses the luciferase gene was expressed under the early etl-promoter (early transcript large) in the same plasmid. both receptor subtypes showed the same rank o ... | 1995 | 8575536 |
| replication of a mamestra brassicae nuclear polyhedrosis virus in a newly established mamestra brassicae cell line. | a continuous cell line, designated mbl-3, was newly established from minced neonate larvae of the cabbage armyworm, mamestra brassicae. this new cell line is heteroploid, containing triploid cells predominantly at the frequency of about 50%. doubling time of the cell population is 33 hrs. the akutsu isolate of a mamestra brassicae nuclear polyhedrosis virus (mbnpv) was examined for replication in 16 continuous cell lines, including the cell line mbl-3, from seven lepidoptera species: mamestra br ... | 1995 | 8578995 |
| improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection. | the extracellular domain of the epidermal growth factor receptor (egfr) was expressed using the baculovirus expression vector system. the maximum level of the egfr extracellular domain secreted into the medium in sf-9 (spodoptera frugiperda or fall army-worm) cell batch culture was approximately 2.5 micrograms ml-1. in order to increase this yield, a process was developed that included the following sequence of steps: batch growth to maximum cell density, infection of the cells with recombinant ... | 1995 | 8579836 |
| a viral movement protein as a nuclear shuttle. the geminivirus br1 movement protein contains domains essential for interaction with bl1 and nuclear localization. | for the nuclear replicating bipartite geminiviruses such as squash leaf curl to systemically infect the host requires the active participation of two virus-encoded movement proteins, br1 and bl1. these act in a cooperative manner to transport the viral single-stranded dna genome from its site of replication in the nucleus to the cell periphery (a.a. sanderfoot, s.g. lazarowitz [1995] plant cell 7: 1185-1194). we have proposed that br1 functions as a nuclear shuttle protein, transporting the vira ... | 1996 | 8587985 |
| induction of a metabolic switch in insect cells by substrate-limited fed batch cultures. | insect cell metabolism was studied in substrate-limited fed batch cultures of spodoptera frugiperda (sf-9) cells. results from a glucose-limited culture, a glutamine-limited culture, a culture limited in both glucose and glutamine, and a batch culture were compared. a stringent relation between glucose excess and alanine formation was found. in contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. simultaneous glucose and g ... | 1995 | 8590651 |
| evidence for a proprotein intermediate during maturation of type ii antifreeze protein in sea raven, hemitripterus americanus. | the circulating type ii antifreeze protein (afp) in sea raven is 129 amino acids (aa) long (14 kda) and is derived from an initial 163 aa translation product that is synthesised in the liver. signal peptide cleavage algorithms, as well as transgenic expression studies in fall armyworm cells, predict the formation of a 146 aa (16 kda) proprotein intermediate. a protein of this size that cross-reacted with anti-sea raven afp antibody was detected in sea raven serum using phosphate/urea sds-page, a ... | 1996 | 8597578 |
| human beta2-glycoprotein i as an anticardiolipin cofactor determined using mutants expressed by a baculovirus system. | beta2-glycoprotein i (beta2-gpi) consists of five repeats of a homologous domain. we designed a series of human beta2-gpi mutant genes, ie, three mutant genes lacking the domain(s) present in the nh2-terminal region and two of those present in the cooh-terminal region. these mutant genes were expressed in spodoptera frugiperda insect cells (sf9) infected with recombinant baculoviruses and the mutant proteins were secreted into the culture medium. the molecular mass of the purified mutant protein ... | 1996 | 8605342 |
| actin binding and proteolysis by the baculovirus acmnpv: the role of virion-associated v-cath. | infection of larvae by autographa californica m nuclear polyhedrosis virus (acmnpv) results in liquefaction of susceptible hosts, presumably due to the breakdown of cells and extracellular matrices. in spodoptera frugiperda tissue culture cells, infection leads to dramatic rearrangement and eventual destruction of the actin cytoskeleton. the first of these rearrangements is the formation of actin cables in the cytoplasm of the cell. cable formation requires release of the budded virus (bv) nucle ... | 1996 | 8607267 |
| a diagnostic immunoassay for newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus. | a recombinant baculovirus containing a cdna clone encoding the nucleocapsid (np) protein of newcastle disease virus (strain ulster 2c) has been used to infect insect cells (spodoptera frugiperda). high levels of overexpressed np protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect elisa to detect the presence of anti-ndv antibo ... | 1995 | 8609201 |
| the dna polymerase and helicase genes of a baculovirus of orgyia pseudosugata. | regions of the genome of the orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (opmnpv) containing the dna polymerase and helicase genes were sequenced. the dna polymerase and helicase genes encode predicted proteins of 985 (112.6 kda) and 1223 (140.5 kda) amino acids and exhibited 63 % and 59 % amino acid identity, respectively, with their homologues in the autographa californica mnpv (acmnpv). the influence of sequence variation between the opmnpv and acmnpv dna polymerase and ... | 1996 | 8609478 |
| induction of specific allograft immunity by soluble class i mhc heavy chain protein produced in a baculovirus expression system. | spodoptera frugiperda (sf9) insect cells secreted a class i mhc rt1.aa heavy chain protein when infected with baculovirus that bore a construct that contained a honeybee melittin secretion (ms) signal attached to rt1.aa cdna. the rt1.aa heavy chain protein in the culture supernatant and cell lysate immunoprecipitated in the presence of 5 individual anti-rt1.aa-specific mab. as was revealed by densitometric analysis, the ms signal increased the production (7- to 17-fold) and secretion (20- to 47- ... | 1996 | 8610360 |
| studies of choristoneura fumiferana nuclear polyhedrosis virus gene expression in insect cells. | to investigate the mechanisms regulating baculovirus virulence and host range we have begun to study choristoneura fumiferana nuclear polyhedrosis virus (cfmnpv) and its gene expression in permissive and nonpermissive cells. we have identified and mapped three genes on the cfmnpv genome. the polyhedrin gene is located from 0.0 to 2.0 m.u. and two other genes, dnapol and p143, both of which are essential for baculovirus dna replication, are located from 35.3 to 40.9 m.u. and 55.5 to 63.4 m.u., re ... | 1996 | 8610448 |
| purification of a virus-induced rna polymerase from autographa californica nuclear polyhedrosis virus-infected spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro. | the virus-induced rna polymerase from autographa californica nuclear polyhedrosis virus-infected spodoptera frugiperda cells was separated from the three host nuclear rna polymerases by deae-sephadex chromatography and then purified through two more steps: heparin-agarose chromatography and glycerol gradient ultracentrifugation. fractions from each of these purification steps have been assayed in vitro for the ability to perform accurate initiation of transcription on a late (p6.9) and a very la ... | 1996 | 8614978 |
| trans-activation of a cell housekeeping gene promoter by the ie1 gene product of baculoviruses. | protein ie1 is the product of a baculovirus gene, ie1, that is activated immediately upon entrance of the viral genome into the cell nucleus. this protein was previously shown to be a trans-regulator of viral genes whose products are required for initiation of the infectious cycle including viral dna replication. to test whether the ie1 protein is also capable of trans-regulating nuclear genes of the host in vitro and in vivo, we transfected the ie1 gene of bombyx mori nuclear polyhedrosis virus ... | 1996 | 8615012 |
| the role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity. | the microsomal triacylglycerol transfer protein (mtp), an alpha beta dimer, is obligatory for the assembly of apob-containing lipoproteins in liver and intestinal cells. the beta subunit is identical with protein disulphide isomerase, a 58 kda endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. we report here the expression of the human mtp subunits in spodoptera frugiperda cells. when the alpha subunit was expressed alone, ... | 1996 | 8615825 |