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1.
Whereas bacterial expression systems are widely used for production of uniformly or selectively 15N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively 15N-labeled proteins in insect cells. The quantities of 15N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the 15N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.  相似文献   

2.
A secretory form of human α3-fucosyltransferase IX (sFUT9) was overexpressed in Spodoptera frugiperda (Sf9) insect cells using the stable expression vector pIB/V5-His-TOPO and the signal sequence of human interleukin 2 for efficient secretion. sFUT9 was active and its three potential N-glycosylation sites were occupied. sFUT9 efficiently fucosylated the type II acceptors Galbeta4GlcNAC-R and Fucalpha2Galbeta4GlcNAc-R (R = (CH2)3NHCO(CH2)5–NH-biotin) but not the corresponding sialylated acceptor, and only very poorly the type I (Galbeta3GlcNAc-R) related acceptors. sFUT9 showed a clear preference for glycoproteins containing type II acceptors, with values of 121, 113 and 110 microU/million cell for asialofetuin, erythropoietin and asialoerythropoietin, respectively, values approximately 11-fold higher than those obtained for the small acceptors.  相似文献   

3.
Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.  相似文献   

4.
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 different multiplicities of infection: the timing and maximum level of expression of the recombinant protein were dependent on the multiplicity of infection, the cell density at infection, and on the aeration rate of the culture. Oxygen-limited growth resulted in undetectable levels of recombinant protein (< 6 ng recombinant protein 10(-7) cells). Compared with the maximum yields observed in spinner flask cultures, higher levels of recombinant protein were produced when cells were grown and infected in the bioreactor. The level of dissolved oxygen in the bioreactor was controlled at 50% of air saturation.  相似文献   

5.
The toxicity of sixteen fungal metabolites produced by some entomopathogenic fungi or biological control fungi agents was evaluated on lepidopteran Spodoptera frugiperda (SF-9) cell line by Trypan blue dye exclusion and MTT-colorimetric assay, after 48 h of incubation. No statistical difference was found between IC50values (50% Inhibiting Concentration) and CC50 values (50% Cytotoxicity Concentration) obtained by MTT test and Trypan blue dye exclusion for each fungal metabolite. By MTT assay, the cytotoxicity ranking was fusarenon X (IC50 0.3 microM) = diacetoxyscirpenol (IC50 0.5 microM) = beauvericin (IC50 2.5 microM) = nivalenol (IC50 5.3 microM) = enniatin (IC50 6.6 microM) > or = gliotoxin (IC50 7.5 microM) > zearalenone (IC50 17.5 microM) > deoxynivalenol (IC50 47.6 microM). By Trypan blue dye exclusion the cytotoxicity ranking was fusarenon X (CC50 0.4 microM) = diacetoxyscirpenol (CC50 1.1 microM) beauvericin = (CC50 3.0 microM)=gliotoxin (CC50 4.0 microM) = enniatin (CC50 6.7 microM) > or = nivalenol (CC50 9.5 microM) > zearalenone (CC50 18.3 microM) > deoxynivalenol (CC50 45.0 microM). The comparison with other bioassays showed that the SF-9 insect cell line could represent a further tool to screen for the toxic effects of fungal metabolites especially for beauvericin, gliotoxin, and zearalenone.  相似文献   

6.
The effects of calcium ions and modulators of calcium movement on Bacillus thuringiensis insecticidal protein toxicity were investigated with Sf9 cells (Spodoptera frugiperda, fall armyworm) by a new B. thuringiensis toxicity assay based on measurement of fluorescence of ethidium homodimer, a high-affinity DNA stain. CryIC toxicity was substantially stimulated by extracellular calcium in a dose-dependent way (in the millimolar range), while toxicity enhancement could not be replicated when calcium was replaced by barium. This incremental toxicity was reduced by cobalt and lanthanum ions, two inorganic-calcium transport inhibitors. Methoxyverapamil, a voltage-dependent calcium channel blocker, and nifedipine, an inhibitor of dihydropyridine-sensitive L-type calcium channels, had no effect on CryIC toxin activity, but BAY K 8644, an L-type calcium channel activator, increased CryIC activity at high concentrations of extracellular calcium. While A23187, a calcium ionophore, and TMB-8, an inhibitor of intracellular-calcium mobilization, did not change CryIC-induced mortality, thapsigargin, an inhibitor of calcium uptake in intracellular stores, and more particularly trifluoperazine, which inhibits calcium-calmodulin-dependent processes, increased CryIC-mediated toxicity. The incremental effect of extracellular calcium on CryIC-induced toxicity was consistent with an increased concentration of intracellular calcium.  相似文献   

7.
Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of approximately 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R-(-)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands.  相似文献   

8.
The goal of this research was to induce neuron-like properties in Sf21 cells, an insect ovarian cell line, which could lead to a new high-throughput insecticide screening method and a way to mass produce insect neuronal material for basic research. This study applied differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, a maximum of ca. 30?% of Sf21 cells expressed an apparent neuronal morphology of unipolar, bipolar, or multipolar axon-like processes within 2?C3?days. Maximal differentiation occurred after 2?days in the presence of 50???M 20-HE or 3?days in 10???M insulin. Both 20-HE and insulin displayed time- and concentration-dependent differentiation with biphasic curves, suggesting that two binding sites or processes were contributing to the observed effects. In addition, combinations of 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system stimulant, inhibited induction of elongated processes by 20-HE and/or insulin, with an IC50 of 9 nM for 20-HE, and the inhibition was incomplete, resulting in about one-quarter of the differentiated cells remaining, even at high concentrations (up to 1?mM). The ability to induce a neural phenotype simplifies the studies of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined.  相似文献   

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11.
L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1-Ig6), five fibronectin-type III (FN1-FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily glycosylated in a variety of cell types. In this work, seven truncated soluble forms including L1 ectodomain (L1/ECD) and Ig domains 5-6 (L1/Ig5-6) have been constructed by PCR and have been cloned, as well as the full-length form (L1), in the stable expression vector for insect cells pMIB/V5-His-TOPO. Spodoptera frugiperda Sf9 cell lines expressing the truncated forms have been obtained, and all proteins were successfully secreted. L1/ECD and L1/Ig5-6 were produced in shake flasks with productions of 3 and 32 mg/L on the third and fourth day of culture, respectively. When L1/Ig5-6 was produced for four days in 2L bioreactor 200 mg/L protein were recovered from the supernatants on the fourth day of culture. Affinity-purified L1/ECD and L1/Ig5-6 were immobilized on poly-d-lysine coated coverslips, and were shown to be active in inducing neurite outgrowth from human NT2N neurons. Therefore, correctly folded and functional truncated forms of human L1 have been produced in high amounts from insect cells using a stable expression system.  相似文献   

12.
The scarcity of fundamental knowledge on the baculovirus-host cell interaction is a major drawback for the improvement of bioprocesses through Metabolic Engineering. After the first hours post-infection, the virus takes over the control of cellular machinery, leading to the repression of host gene expression and imposing a high metabolic burden to insect cells. Nevertheless, there is a lack of detailed data on the metabolic responses to infection, which are ultimately responsible for system productivity performance. In this work, a further insight into the central metabolism of Sf9 cells is achieved by a combined analysis of enzyme activities, cellular cofactors (ATP and NAD(P)(+)/NAD(P)H) and metabolic fluxes. Hexokinase and isocitrate dehydrogenase were identified as feasible limiting steps of metabolism; carbon and nitrogen metabolism enzymes were differentially regulated during batch cultures. Moreover, alterations occurring after infection demonstrated the importance of maintaining the energetic state of the cells for baculovirus replication, since ATP accumulated in a MOI-dependent way, and the glutamate dehydrogenase anaplerotic pathway was greatly activated. Altogether, cellular de-energization and stress responses are relevant factors in the metabolic burden imposed by infection. The implications for the improvement of bioprocess performance are discussed.  相似文献   

13.
This is the second of two papers [Drews, M., Doverskog, M., Ohman, L., Chapman, B.E., Jacobsson, U., Kuchel, P.W., H?ggstr?m, L., 2000. Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR. J. Biotechnol. 78, 23-37]. where the general goal has been to determine and characterise the glutamine metabolism in Sf9 cells. The presence of glutamate synthase (GOGAT) activity was investigated in cell-free extracts of S. frugiperda (Sf9) insect cells by modified 1H/15N spin-echo and gradient enhanced multiple quantum coherence NMR spectroscopy techniques. Cell-free extracts were prepared from cells cultured in a serum-free medium. The assay conditions were based on conventional spectrophotometric and chromatographic methods. NMR data showed that nitrogen from [5-15N] glutamine was selectively incorporated into 2-oxoglutarate forming [2-15N] glutamate with a specific activity of 4.15 +/- 0.21 nmol [2-15N] glutamate min -1 (mg total protein)-1 in the cell-free extracts. The enzyme activity was exclusively dependent on NADH as coenzyme and was completely inhibited by 1 mM azaserine. From the results obtained, we conclude that Sf9 cells possess NADH-GOGAT activity. Furthermore, the high specificity of the NMR method enables distinction of competing reactions from glutaminase and glutamate dehydrogenase.  相似文献   

14.
The sex steroid binding protein (SBP) which binds androgens circulating in the blood of man has been examined to determine the structural requirements for high affinity binding. SBP was purified partially and the ability of each of more than 150 steroids to compete with [3H]dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) for binding to SBP was assessed.Binding was enhanced by reduction of the Δ4 double bond to 5α-dihydro, addition of a methyl group at C-4 and in one case unsaturation at C-14, 15. Affinity was always reduced by modifications of the C-17β hydroxy. Binding was also severely decreased by deletion of the keto moiety at C-3; however, relatively high affinity was retained by an alcohol or an unsubstituted pyrazole group at C-3. Certain alpha surface substitutions such as 17α-ethinyl had limited effects on binding; whereas, other modifications such as 7α-methyl or 17α-methyl caused significant reduction in binding. Most modifications at C-2, 6, 9 or 11 also impaired affinity, and the 5β steroids had reduced affinity.  相似文献   

15.
A coupled cell-free translation/glycosylation system, prepared from Spodoptera frugiperda insect cells, was established and optimized for protein production and glycosylation efficiency. Both translation and glycosylation were stimulated by addition of Mg2+, K+, ATP, GTP, creatine kinase and creatine phosphate, suggesting that glycoprotein productivity is largely determined by translation efficiency. However, high concentrations of creatine phosphate significantly inhibited translation. Spermidine stimulated both translation and glycosylation, but glycosylation required higher concentrations of spermidine than translation. Furthermore, extracts prepared at a nitrogen pressure of 10 kg/cm2 with the Mini-Bomb cell disruption chamber had the highest glycoprotein productivity; and extracts prepared at the higher nitrogen pressure of 15 kg/cm2 retained glycosylation ability. While extracts prepared with the Potter-Elvehjem homogenizer could mediate translation, no glycosylation was achieved. This indicated that the posttranslational machinery might survive disruption by high pressure, but not by physical shearing force. This insect cell-free system was able to synthesize approximately 25 microg of glycosylated gp120/ml of reaction mixture.  相似文献   

16.
Spodoptera frugiperda (Sf9)-cells differ markedly in their proteinglycosylation capacities from vertebrate cells in that theyare not able to generate complex type oligosaccharide side chains.In order to improve the oligosaccha ride processing propertiesof these cells we have used baculovirus vectors for expressionof human (ß1,2-N-acetylglucosaminyltransferase I (hGNT-I),the enzyme catalysing the crucial step in the pathway leadingto complex type N-glycans in vertebrate cells. One vector (Bac/GNT)was designed to express unmodified GNT-I protein, the secondvector (Bac/tagGNT) to express GNT-I protein with a tag epitopefused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virusa protein of about 50 kDa representing hGNT-I was detected withan antiserum directed against the tag epitope. HGNT-I activitywas increased at least threefold in lysates of infected cellswhen N-acetylglucosamine (GlcNAc)-free ovalbumine was used assubstrate. To monitor hGNT-I activity in intact Sf9-cells, theglycosylation of coexpressed fowl plague virus hemagglutinin(HA) was investigated employing a galactosylation assay andchromatographic analysis of isolated HA N-glycans. Coexpressionof hGNT-I resulted in an at least fourfold increase of HA carryingterminal GlcNAc-residues. The only structure detectable in thisfraction was GlcNAcMan3GlcNAc2. These results show that hGNT-Iis functionally active in Sf9-cells and that the N-glycans ofproteins expressed in the baculovirus/insect cell system areelongated by coexpression of glycosyltransferases of vertebrateorigin. Complete complex type oligosaccharide side chains werenot observed when hGNT-I was overexpressed, thus supportingthe concept that Sf9-cells do not contain glycosyltransferasesacting after hGNT-I. ß1,2-N-acetylglucosaminyltransferase I baculovirus expression of recombinant protiens N-glycosylation in Sf9-cells  相似文献   

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MOTIVATION: A whole set of Expressed Sequence Tags (ESTs) from the Sf9 cell line of Spodoptera frugiperda is presented here for the first time. By this way we want to identify both conserved and specific genes of this pest species. We also expect from this analysis to find a class of protein sequences providing a tool to explore genomic features and phylogeny of Lepidoptera. RESULTS: The ESTs display both housekeeping as well as developmentally regulated genes, and a high percentage of sequences with unknown function. Among the identified ORFs, almost all ribosomal proteins (RPs) were found with high EST redundancy and hence sequence accuracy. The codon usage found among RP genes is in average surprisingly much less biased in Lepidoptera than in other organisms. Other Spodoptera genes also displayed a low bias, suggesting a general genome expression feature in this Lepidoptera. We also found that the L35A and L36 RP sequences, respectively, display 40 and 10 amino-acid insertions, both being present only in insects. Sequence analysis suggests that they are probably not subjected to a strong selective pressure and may be good phylogenetic markers for Lepidoptera. Most interestingly, the Lepidoptera sequences of 9 RP genes displayed a specific signature different from the canonical one. We conclude that the RP family allows valuable comparative genomics and phylogeny of Lepidoptera. AVAILABILITY: All EST sequence data are available from the private 'Spodo-Base' upon request.  相似文献   

19.
The DNA coding for the circumsporozoite protein (CPS) of Plasmodium falciparum has been cloned into the baculovirus expression vector pAcYM1 and expressed in Spodoptera frugiperda (Sf9) insect cells. Three DNA constructs have been made: the first one directs the synthesis of the complete CSP (aa 1–412), the second leads to the production of a species devoid of the anchor domain (aa 1–391) and the third one to a molecule lacking both signal and membrane anchor sequences (aa 18–391). All three recombinant CPS were produced at about 3 g per 106 infected cells and were characterized in terms of immunoreactivity and apparent molecular weight. Analytical purification of the recombinant proteins was achieved by a combination of heat treatment, acidification, isoelectric focusing and ion exchange chromatography. The purified material, when injected into mice, generated only modest antibody responses, although antisera from immunized mice reacted with control CSP antigens carrying or not the major immunodominant repeat region.Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus - CSP circumsporozoite protein  相似文献   

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