• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.200-203
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• 1994

Spodoptera frugiperda IPLB-SF-21-AE cells were cultivated in a spin-filter bioreactor with continuous perfusion for the recombinant $\beta$-galactosidase production. At the perfusion rate of 0.06 $hr^{-1}$, the maximum cell density of insect cells in this bioreactor system reached 3.5$\times$$l0^6$ viable cells/ml using the Grace media containing 5% FBS and 0.3% Pluronic F-68. The recombinant $\beta$-galactosidase production of 8, 100 units per reactor volume was also achieved at this perfusion rate.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.53-56
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• 1996

To investigate the molecular biological marker in insect cells, BmN-4 and Sf-0 cells were analysed by SDS-PAGE and random amplification of polymorphic DNA. The results showed that the patterns of total cell protein and random amplification of polymorphic DNA were distinguished between BmN-4 and Sf-9 cells, suggesting that the unique major bands were useful as molecular biological marker in BmN-4 and Sf-9 cells.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.183-190
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• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• Biomedical Science Letters
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• v.15 no.1
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• pp.55-60
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• 2009

Insect cells such as Spodoptera frugiperda Clone 9 (Sf9) cells are widely chosen as the host for heterologous expression of a mammalian sugar transport protein using the baculovirus expression system. Characterization of the expressed protein is expected to include assay of its function, including its ability to transport sugars and to bind inhibitory ligands such as cytochalasin B. It is therefore very important first to establish the transport characteristics and other properties of the endogenous sugar transport proteins of the host insect cells. However, very little is known of the transport characteristics of Sf9 cells, although their ability to grow on TC-100 medium strongly suggested the presence of endogenous glucose transport system. In order to investigate the substrate and inhibitor recognition properties of the Sf9 cell transporter, the ability of pentoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. To determine the time period over which of sugar into the Sf cells was linear, the uptake of 2dGlc 0.1mM extracellular concentration was measured over periods ranging from 30 seconds to 30 minutes. The uptake was linear for at least 2 minutes at the concentration, implying that uptake made over a 1 minute time course would reflect initial rates of the sugar uptake. The data have also revealed the existence of a saturable transport system for pentose uptake by the insect cells. The transport was inhibited by D-xylose and D-ribose, although not as effective as hexoses. However, L-xylose had a little effect on 2dGlc transport in the Sf9 cells, indicating that the transport is stereoselective. Unlike the human erythrocyte-type glucose transport system, D-ribose had a somewhat greater apparent affinity for the Sf9 cell transporter than D-xylose. It is therefore concluded that Sf9 cells contain an endogenous sugar transport activity that in some aspects resembled the human erythrocyte-type counterpart, although the Sf9 and human transport systems do differ in their affinity for cytochalasin B.

• Biomedical Science Letters
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• v.11 no.1
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• pp.57-61
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• 2005

The number of sites at which a protein can be readily cleaved by a proteolytic enzyme is greatly influenced by its three-dimensional structure. For native, properly-folded proteins both the rate of cleavage and number of sites at which cleavage takes place are usually much less than for the denatured protein. In order to compare the tertiary structure of recombinant HepG2 type glucose transporter with that of its native counterpart in the erythrocyte, the pattern of tryptic cleavage of the protein expressed in insect cell membranes was therefore examined. After 30 minutes digestion, a fragment of approximate Mr 19,000-21,000 was generated. In addition to this, there were two less intensely stained fragments of apparent Mr 28,000 and 17,000. The pattern of labelling was similar up to 2 hours of digestion. However, the fragments of Mr 19,000-21,000 and Mr 17,000 were no longer detectable after 4 hours digestion. The observation of a very similar pattern of fragments yielded by tryptic digestion of the HepG2 type transporter expressed in insect cells suggests that the recombinant protein exhibits a tertiary structure similar if not identical to that of its human counterpart. Also, the endogenous sugar transporter(s) present in Sf21 cells did not bind cytochalasin B, the potent transporter inhibitor. Therefore, the baculovirus/Spodoptera frugiperda (Sf) cell expression system could be very useful for production of large amounts of human glucose transporters, heterologously.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.122-128
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• 1992

Post-translational modifications of the nucleocapsid protein of bovine coronavirus (Quebec strain) were investigated. Coronavirions were radiolabelled in vivo with inorganic $[^{32}P]$orthophosphate and analysed by SDS-PAGE, followed by autoradiography. A single polypeptide with a migration rate of 55 KDa was identified by metabolic phosphate labelling, demonstrating that the nucleocapsid protein of bovine coronavirus was a phosphoprotein. A gene encoding the nucleocapsid protein was inserted immediately downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis baculovirus. Spodoptera frugiperda cells infected with this recombinant baculovirus synthesized a 55 KDa polypeptide, as demonstrated by immunoprecipitation with anti-nucleocapsid monoclonal antibody. The recombinant nucleocapsid protein synthesized in Spodoptera cells could also be labelled by $[^{32}P]$orthophosphate. Phosphoamino acid analysis showed that both serine and threonine residues were phosphorylated in authentic, as well as in recombinant nucleocapsid proteins, with a relative phosphorylation ratio of 7:3. Our studies demonstrated that the nucleocapsid protein of bovine coronavirus was a serine and threonine-phosphorylated protein and that Spodoptera insect cells were able to properly phosphorylate the relevant foreign proteins.

• Biomedical Science Letters
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• v.9 no.4
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• pp.177-181
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• 2003

The baculovirus/Spodoptera frugiperda (Sf) cell system has become popular for the production of large amounts of the human erythrocyte glucose transporter, GLUT1, heterologously. However, it was not possible to show that the expressed transporter in insect cells could actually transport glucose. The possible reason for this was that the activity of the endogenous insect glucose transporter was extremely high and so rendered transport activity resulting from the expression of exogenous transporter very difficult to detect. Sf21-AE cells are commonly employed as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains 0.1 % D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike the human glucose transporter, very little is known about properties of the endogenous sugar transporter(s) in insect cells. Thus, the uptake of 2-deoxy-D-glucose (2dGlc) by Sf21-AE cells and the inhibition of 2dGlc transport in the insect cells by fructose and cytochalasin B were investigated in the present work. The binding assay of cytochalasin B was also performed, which could be used as a functional assay for the endogenous glucose transporter(s) in the insect cells. Sf21-AE cells were infected with the recombinant virus AcNPV-GT or no virus, at a multiplicity of infection (MOI) of 5. Infected cells were resuspended in PBS plus and minus 300 mM fructose, and plus and minus 20 $\mu$M cytochalasin B for use in transport assays. Uptake was measured at 28$^{\circ}C$ for 1 min, with final concentration of 1 mM deoxy-D-glucose, 2-[1,2-$^3$H]- or glucose, L-[l,$^3$H]-, used at a specific radioactivity of 4 Ci/mol. The results obtained demonstrated that the sugar uptake in uninfected cells was stereospecific, and was strongly inhibited by fructose but only poorly inhibitable by cytochalasin B. It is therefore suggested that the Sf21-AE glucose transporter has very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• Biomedical Science Letters
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• v.11 no.4
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• pp.487-492
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• 2005

The baculovirus/insect cell expression system is of great value in the study of structure-function relationships in mammalian glucose-transport proteins by site-directed mutagenesis and for the large-scale production of these proteins for mechanistic and biochemical studies. Spodoptera frugiperda Clone 21 (Sf2l) cells grow well on TC-100 medium that contains $0.1\%$ D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, very little is known about the properties of the endogenous sugar transporter(s) in Sf2l cells, although a saturable transport system for hexose uptake has been previously revealed in the Sf cells. In order to further examine the substrate and inhibitor recognition properties of the Sf2l cell transporter, the ability of hexoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. The $K_i's$ for reversible inhibitors were determined from plots of uptake versus inhibitor concentration. Transport was effectively inhibited by D-mannose and D-glucose. Of the hexoses tested, L-glucose had the least effect on 2dGlc transport in the Sf2l cells, indicating that the transport is stereoselective. Unlike the human HepG2 type glucose transport system, D-mannose had a somewhat greater affinity for the Sf2l cell transporter than D-glucose, implying that the hydroxyl group at the C-2 position is not necessary for strong binding. However, epimerization at the C-4 position of D-glucose (D-galactose) resulted in a dramatic decrease in affinity of the hexose for the Sf2l cell transporter. Such a lowering of affinity might be the result of the involvement of the C-4 hydroxyl in hydrogen bonding. It is therefore suggested that Sf2l cells were found to contain an endogenous sugar transport activity that in several aspects resembles the human HepG2 type glucose transporter, although the insect and human transporters do differ in their affinity for cytochalasin B.

• BMB Reports
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• v.31 no.5
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• pp.453-458
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• 1998

Human thrombopoietin (hTPO) was expressed to high levels in insect cells using the baculovirus expression system. Full-length hTPO cDNA containing a native signal peptide sequence was amplified by PCR from a human fetal liver cDNA library and cloned into the Autographa californica nuclear polyhedrosis virus (AcNPV) expression vector. Immunoblot analysis with antiserum against hTPO indicated that an approximately 55 kDa protein was produced in recombinant AcNPV infected insect cells. Recombinant hTPO was produced 4-fold higher in Trichoplusia ni (Tn5) cells than in Spodoptera frugiperda (Sf9) cells. with most of the hTPO produced in Tn5 cells secreted into the culture medium. Addition of tunicamycin in the culture medium resulted in the reduction of the size of hTPO to 35-38 kDa, and most of the protein remained within the cell. These results suggest that N-glycosylation of hTPO is required for the secretion of the protein into the culture medium in insect cells. hTPO produced in insect cells induced proliferation and maturation of megakaryocyte progenitors, indicating that it is in a biologically active form.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.355-362
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• 2000

The Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) gene in the pHLA-21 plasmid was inserted into a baculovirus (Hyphantria cunea nuclear polyhedrosis virus) expression vector (lacZ-HcNPV) to construct a recombinant virus gB-HcNPV expressing gB. Spodoptera frugiperda cells infected with this recombinant virus synthesized and processed gB of approximately 120 kDa, which cross-reacted with the monoclonal antibody to gB. The recombinant gB was identified on the membrane of the insect cells using an immunofluorescence assay. Antibodies to this recombinant raised in mice recognize the viral gB and neutralized the infectivity of the HSV-1 in vitro. These results show that the gB gene has the potential to be expressed in insect cells. They also demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the lacZ-HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV. Accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.