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PubMed1998

Characterization of the human gonadotropin-releasing hormone receptor heterologously produced using the baculovirus/insect cell and the Semliki Forest virus systems.

1. Two eukaryotic viral systems, the baculovirus/insect cell and the Semliki Forest virus systems, were tested for heterologous expression of human gonadotropin-releasing hormone receptor (GnRHR) cDNA. 2. An unmodified as well as a c-myc epitope-tagged human GnRH receptor was produced in two insect cell lines (Spodoptera frugiperda, Trichoplusia ni) after infection with the respective recombinant baculoviruses. In both insect cell lines, the receptor was identified by immunoblot analysis as a triplet of bands between 35 and 40 kDa. After deglycosylation of the receptor the molecular mass decreased to 35 kDa. The GnRH receptor was localized in membrane compartments within the infected insect cells. However, only in membranes of infected Trichoplusia ni insect cells could approximately 2000 receptors per cell be detected. 3. Production of the GnRH receptor in BHK cells using the Semliki Forest virus system resulted in approximately 50,000 receptors per cell. A maximal yield of 0.42 pmol/mg membrane protein was obtained 24 hr after electroporation of BHK cells with in vitro synthesized RNA. Binding of the antagonist [125I]Cetrorelix was saturable with a KD of 1.3 nM. The receptor produced in the BHK cells was further characterized by ligand displacement studies. The rank order of agonist and antagonist affinities was Cetrorelix > Triptorelin > Antide > GnRH.

Amino Acid Sequence↗

PubMed2004

Expression and enzyme activity determination of human cyclooxygenase-1 and -2 in a baculovirus-insect cell system.

AIM: To develop an in vitro intact cell-based assay for screening selective cyclooxygenase inhibitors. METHODS: Human cyclooxygenase-1 (hCOX-1) and cyclooxygenase-2 (hCOX-2) genes were cloned from human monocyte cell line THP-1 cells and expressed in Spodoptera frugiperda (sf9) insect cell line by Bac-to-Bac baculovirus expression systems. Infected sf9 cells were harvested 24 h post-infection (hpi), and distributed to a 24-well plate, preincubated with various nonsteroidal anti-inflammatory drugs, and challenged with 10 mmol/L arachidonic acid; the cyclooxygenase activity was assessed indirectly by prostaglandin E2-specific radioimmunoassay. RESULTS: Polymerase chain reaction detection demonstrated that hCOX-1 and hCOX-2 were transposed to the bacmid. Western blot analysis showed that infected sf9 cells could express hCOX-1 and hCOX-2 proteins. Radioimmunoassay demonstrated that both recombinant proteins functioned well in sf9 cells. CONCLUSION: Human cyclooxygenase-1 and cyclooxygenase-2 were successfully expressed in sf9 insect cell line. It can be utilized for the identification of potent and selective inhibitors of hCOX-1 and/or hCOX-2.

Animals↗

PubMed2006

Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce "mammalianized" recombinant glycoproteins.

Baculovirus expression vectors are frequently used to express glycoproteins, a subclass of proteins that includes many products with therapeutic value. The insect cells that serve as hosts for baculovirus vector infection are capable of transferring oligosaccharide side chains (glycans) to the same sites in recombinant proteins as those that are used for native protein N-glycosylation in mammalian cells. However, while mammalian cells produce compositionally more complex N-glycans containing terminal sialic acids, insect cells mostly produce simpler N-glycans with terminal mannose residues. This structural difference between insect and mammalian N-glycans compromises the in vivo bioactivity of glycoproteins and can potentially induce allergenic reactions in humans. These features obviously compromise the biomedical value of recombinant glycoproteins produced in the baculovirus expression vector system. Thus, much effort has been expended to characterize the potential and limits of N-glycosylation in insect cell systems. Discoveries from this research have led to the engineering of insect N-glycosylation pathways for assembly of mammalian-style glycans on baculovirus-expressed glycoproteins. This chapter summarizes our knowledge of insect N-glycosylation pathways and describes efforts to engineer baculovirus vectors and insect cell lines to overcome the limits of insect cell glycosylation. In addition, we consider other possible strategies for improving glycosylation in insect cells.

Animals↗

PubMed2006

Oligosaccharides modulate the apoptotic activity of glycodelin.

GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitor of sperm-zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insect cell line yielded an apoptotically active Gd; however, the same gene expressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary (CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans.

Animals↗

PubMedDate not supplied

Production of recombinant proteins by baculovirus-infected gypsy moth cells.

An experimental study was undertaken to evaluate alternative insect cell lines to Sf9 [from Spodoptera frugiperda (fall armyworm)] for the production of recombinant proteins. Insect cell lines from two different organisms were considered: IPLB-LdEIta (LdEIta) from Lymantria dispar (gypsy moth) and IPLB-HvT1 (HvT1) from Heliothis virescens (tobacco budworm). Both LdEIta and HvT1 produced higher total activity levels of recombinant beta-galactosidase in monolayer culture than Sf9 after infection with the Autographa californica nuclear polyhedrosis virus (AcMNPV). However, only LdEIta generated a product yield (activity per milligram of total protein) which exceeded that of Sf9 (by 25%), so its growth and production characteristics were investigated in depth. LdEIta generated production levels and yields of a recombinant rotaviral protein, VP4, which exceeded those of Sf9 by 84 and 38%, respectively. In suspension culture, the LdEIta cells grew as aggregates with a doubling time several hours longer than Sf9, but the recombinant product yields of LdEIta were still higher than Sf9 by 38% in this culture environment. beta-Galactosidase expression rates and cell death rates suggested that the difference in productivity between the two hosts was due to the ability of LdEIta to survive the baculovirus infection and produce recombinant proteins longer than Sf9. The presence of LdEIta aggregates in suspension culture may be used as a method to separate live cells from dead cells, labile product, and spent medium in recombinant protein production processes.

Animals↗

PubMed1993

Effects of baculovirus infection on IE1-mediated foreign gene expression in stably transformed insect cells.

Previously, we produced transformed insect cell lines that can express a selected foreign protein constitutively, in the absence of baculovirus infection (D. L. Jarvis, J. G. W. Fleming, G. R. Kovacs, M. D. Summers, and L. A. Guarino, Bio/Technology 8:950-955, 1990). These cells contain stably integrated copies of chimeric genes consisting of the promoter from an immediate-early baculovirus gene, IE1, and the sequences encoding either human tissue plasminogen activator or Escherichia coli beta-galactosidase. Transcription of the integrated genes in these cells is specifically controlled by the IE1 promoter. The purpose of this study was to determine how baculovirus infection influences IE1-mediated foreign protein production by these stably transformed insect cell lines. The results showed that viral infection transiently stimulated and then strongly inhibited the production of both tissue plasminogen activator, a secreted protein, and beta-galactosidase, an intracellular protein. These effects reflected virus-induced changes in the steady-state levels of RNA produced by the integrated genes. Transient assays showed that expression of the viral IEN gene alone could account for the increased levels of RNA observed early in infection. The precise mechanism accounting for the decreased levels of RNA observed later in infection was not determined. However, we obtained evidence that the native IE1 promoter remains active throughout infection, which suggested indirectly that the integrated IE1 promoter is transcriptionally inactivated at late times of baculovirus infection. Thus, the same promoter behaved quite differently late in infection, depending on its local environment. Neither methylation nor degradation appeared to be responsible for inactivating IE1-mediated expression of the integrated genes. The significance of these results with respect to the baculovirus-host interaction and the practical applications of stably transformed insect cell lines are discussed.

Animals↗

PubMed1998

Expression of heterologous proteins in stable insect cell culture.

Stable transformed insect cell lines have been used for producing many highly processed heterologous proteins. Current research has focused on development of new expression and selection systems, and enhancement of vector stability. Defining the variation of modification and processing capabilities between cell lines will further enhance complex protein production from insect cells.

Animals↗

PubMed1997

Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.

Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.

Animals↗

PubMedDate not supplied

Protective effect of methylcellulose and other polymers on insect cells subjected to laminar shear stress.

The relative sensitivity of two insect cell lines to laminar shear stress was determined, and the protective effect of polymers added to the growth media of two insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), was evaluated. TN-368 and SF-9 cells were found to be equally sensitive to laminar shear stress. Methylcellulose [0.5% (w/v) Dow E4M Methocel] and dextran [4.5% (w/v)] increased the resistance of suspended cells to lysis due to laminar shear stress by factors of up to 76 and 28, respectively, compared to cells in media without additives. It was observed that the protective effect of Pluronic F-68 was concentration-dependent: 0.2% and 0.3% (w/v) F-68 increased the resistance of SF-9 cells to shear stress by factors of 15 and 42, respectively. However, increasing the concentration to 0.5% did not significantly increase the cells' resistance compared to 0.3% (w/v). F-68 at 0.2% only increased the resistance of TN-368 cells by a factor of 6. It is believed that the protection is a result of the polymer adsorbing to the cell membrane. None of the polymer additives tested had a significant effect on SF-9 or TN-368 growth rate.

Animals↗

PubMedDate not supplied

An established cell line from the beetle, Xylotrechus pyrrhoderus (Coleoptera: Cerambycidae).

A continuous cell line has been established from larval fat body tissues of the cerambycid beetle Xylotrechus pyrrhoderus Bates. These cells were cultured in MGM-450 medium. The cell line, designated as XP-1, showed a heterogeneous population consisting of spherical and spindle-shaped cells with some capacity to adhere and a doubling time of 5 d. The chromosome number of the cell line ranged from 18 to 42 with a mode of 20. Isozyme analysis showed that the cells had patterns distinctive from those of other insect cell lines. The cells were sensitive to insect hormones, and when continuously treated with 20-hydroxyecdysone and juvenile hormone, they assumed a floating elongated-spindle shape and became strongly adherent, respectively.

Animals↗

PubMed2001

Cytotoxicity of azadirachtin A in human glioblastoma cell lines.

The neem toxin azadirachtin A exhibits selective toxicity on insects. Despite its well-proven efficacy, the mode of action of this toxin remains obscure. The toxicity on vertebrate cells compared to insect cells is also not well characterized. We have cultivated six human glioblastoma cell lines G-28, G-112, G-60 (TP53 mutant) and G-44, G-62, G-120 (TP53 wild-type) in the presence of 28 microM of azadirachtin. This toxin concentration was chosen because it represents the 25 to 50% lethal dose in the glioma cells. Toxicity was measured in terms of cell proliferation (binucleation index), formation of micronuclei and cell survival. In the TP53 mutant cell lines, azadirachtin reduced the proportion of dividing cells and induced formation of micronuclei. Except for G-44 which showed a decrease in binucleation index, proliferation in the TP53 wild-type cell lines was unaffected by azadirachtin. In the TP53 wild-type cell lines, the decrease in micronuclei frequency is attributed to fewer cells entering mitosis to produce micronuclei. This is also apparent from the low surviving fractions. Cell survival was suppressed by 25-69% in all cell lines. The reduction of cell survival is a clear indication that azadirachtin affects reproductive integrity and cell division. The induction of micronuclei reflects DNA damage. Similar studies on damage induction in insect cell lines could elucidate the processes which precede the antifeedant and antimoulting effects of azadirachtin and other neem toxins in insects.

Cell Survival↗

PubMed2002

BCL-2 does not control programmed cell death in the IPLB-LdFB cell line from the insect Lymantria dispar.

In the insect Lymantria dispar cell line IPLB-LdFB the presence of a Bcl-2-like molecule has been demonstrated. The Western blot analysis performed on the cells incubated with 2-deoxy-D-ribose (dRib), an apoptotic inducer, revealed that, in comparison with the control, the Bcl-2 expression was unaffected. Furthermore, incubation of the insect cells with an anti-Bcl-2 polyclonal antibody inhibited the apoptotic effect induced by dRib, and provoked mitochondrial membrane depolarization without any apoptotic phenomena. Similar behaviour was observed using the K+ ionophore valinomycin. From these findings, we hypothesize that the L. dispar Bcl-2-like protein is essential for maintenance of the mitochondrial membrane potential, but not, as usually thought, for the regulation of programmed cell death.

Animals↗

PubMed1998

Toxicity and binding properties of the Bacillus thuringiensis delta-endotoxin Cry1C to cultured insect cells.

A better understanding of the mode of action of Bacillus thuringiensis delta-endotoxins is needed to develop strategies which may prevent or slow down selection for resistance. We studied the effect of Cry1C on several different cultured insect cell lines by means of toxicity assays, ligand blotting, and toxin binding studies. A clear difference in sensitivity toward Cry1C between the insect cell lines was observed. Spodoptera frugiperda cell line Sf9 was most sensitive, whereas Spodoptera exigua cell lines SeUCR and SelZD2109 showed intermediate sensitivity. Mamestra brassicae (Mb0503) and Drosophila melanogaster (Dm1) cells were the least sensitive as compared to Sf9 cells. Ligand blot analysis of SDS-PAGE size-separated proteins showed that Cry1C specifically binds to a 40-kDa protein in Sf9, SeUCR, and SelZD2109 cells. Cry1Ab does not bind to this protein. The Cry1C-binding protein was not observed in Mb0503 and Dm1 cells, suggesting that the presence of the 40-kDa Cry1C-binding protein is correlated with sensitivity toward Cry1C.

Animals↗

PubMedDate not supplied

Production of functional hepatocyte growth factor (HGF) in insect cells infected with an HGF-recombinant baculovirus in a serum-free medium.

Three insect cell lines, SL-7B cells derived from Spodoptera litura, Sf9, and High Five (Hi-5) cells, were used for the production of pro-hepatocyte growth factor (pro-HGF). Cells were cultured and then infected with a recombinant HGF-containing baculovirus in a serum-free medium. In SL-7B cells, pro-HGF is synthesized and excreted from the cells and late in infection is converted to a heterodimeric form of HGF even when the cells are grown in serum free medium. Conversion of a single-chain form of HGF (pro-HGF) into an HGF heterodimer was unexpected, as pro-HGF is normally cleaved by a serum protease called HGF activator. The proliferation activity of heparin-affinity-purified HGF from serum-free culture supernatant of SL-7B cells is comparable to that obtained from HGF converted by serum proteases, suggesting that SL-7B cells produce a functionally analogous protease to correctly process pro-HGF. This work reports, for the first time, on the feasibility of properly processing pro-HGF to form functional HGF by proteases from invertebrate cells in serum-free media. Avoiding the supplementation of sera provides the advantages of a low production cost, zero contamination of infectious agents from sera, and simple downstream product purification. Experimental results further demonstrate that the conversion of pro-HGF by insect cells is cell-line-dependent, because proteases in Hi-5 or Sf9 cells could not process pro-HGF as efficiently and properly as those in SL-7B cells.

Animals↗

PubMed2005

Improvement of the production of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein using a molecular chaperone-assisted insect-cell-based expression system.

A stable Tn-5B1-4 insect cell line co-expressing the recombinant GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 (GFPuv-beta3GnT2) protein fused to a melittin signal sequence with a lectin-like molecular chaperone, human calnexin (hCNX) or human calreticulin (hCRT), was constructed. The expression of either of these molecular chaperones is under the control of a weak promoter, OpMNPV IE2, while that of GFPuv-beta3GnT2 is under the control of Bombyx mori actin promoter. This co-expression system was compared between two different insect cell-baculovirus expression systems: (1) co-infection of the recombinant baculovirus containing a molecular chaperone (AcNPV-hCNX or -hCRT) with a recombinant baculovirus containing GFPuv-beta3GnT2 fused with the melittin signal sequence (AcNPV-me-GGT); (2) infection of AcNPV-me-GGT to a stably expressing cell line for either hCNX or hCRT. In the co-infection system, the intracellular GFPuv-beta3GnT2 expression level was low because of the improved secretion level ratio of the fusion protein, due to the chaperone expression. In the case of infection to the stably expressing cell line for a chaperone, the extracellular GFPuv-beta3GnT2 expression level was similar to the intracellular expression level. This suggests that the amount of expressed chaperone is not sufficient to process beta3GnT2. On the other hand, the co-expression system produced an extracellular beta3GnT activity of 22-23 mU/mL, which was approximately 3.5- and 11-fold higher than those of the stable expression of the fusion gene without the chaperone and the conventional BES with the addition of protease, respectively. The secretion level ratio of the fusion protein of this system increased to 82%, which was approximately 1.5-fold that of any other expression system investigated thus far. These results indicate that the ratio of the expression level of the target gene to that of the chaperone gene may be an important factor in maximizing the production of a target protein. The molecular-chaperone-assisted expression system using a stably transformed insect cell line offers promising prospects for the efficient production of recombinant secretory proteins in insect cells.

Animals↗

PubMed1994

Cell lines used for the selection of recombinant baculovirus.

Four insect cell lines were used to isolate two recombinant baculoviruses which had the beta-galactosidase (beta-gal) gene for colorimetric assay purposes. Plaque assays were performed using two Trichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and two Spodptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blue beta-gal+ recombinants) formed in the Trichoplusia cells was higher than in the Spodoptera cells. The appearance of Autographa californica NPV polyhedra was also faster in the T. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.

Animals↗

PubMed1999

In vitro host range of Autographa californica nucleopolyhedrovirus recombinants lacking functional p35, iap1 or iap2.

We have examined the host range in different insect cell lines of Autographa californica nucleopolyhedrovirus (AcMNPV) recombinants lacking p35, iap1 or iap2. These genes encode, or are predicted to encode, anti-apoptotic proteins. Abrogation of p35 reduced the ability of AcMNPV to replicate in permissive cell lines derived from Spodoptera frugiperda insects by inducing apoptosis. In semi-permissive cell lines, such as Lymantria dispar and Spodoptera littoralis cells, we observed cytopathic effects after infection with AcMNPV but little virus production. Infection of these cells by AcMNPV lacking p35 resulted in apoptosis. However, p35-deficient viruses were still able to replicate normally in Trichoplusia ni, Mamestra brassicae and Panolis flammea cell lines. Disruption of AcMNPV iap1 and iap2 was found not to affect virus replication in any of the cell lines. It was also possible to disrupt both iap1 and iap2 in the same virus without loss of infectivity. A virus without iap1 and p35 demonstrated identical growth characteristics and host range to a virus lacking p35. We conclude that in cells which respond to AcMNPV infection by initiating programmed cell death, the p35 gene product alone is sufficient to inhibit apoptosis. Removal of iap1 or iap2 has no effect on virus replication, even in cell lines which do not undergo apoptosis in response to AcMNPV infection. Our results with two semi-permissive cell lines further indicate that whilst p35 is important in blocking block apoptosis, other factors are involved in restricting AcMNPV replication within these cells.

Animals↗

PubMed1999

The use of bi-cistronic transfer vectors for the baculovirus expression system.

In this communication, we describe the construction of bi-cistronic transfer vectors for the baculovirus expression system (BVES), which are advantageous over the existing vectors. The new vectors provide a simple way to isolate recombinant viruses. More specifically, the gene of interest and the reporter gene luciferase (LUC), constitute the first and second cistrons, respectively, of the same transcript. Therefore, the LUC activity measured during infection of such a bi-cistronic virus, permits an on-line estimation of the recombinant protein level, a very useful feature for large-scale production of recombinant proteins. To achieve expression of the second cistron, the internal ribosome entry site (IRES) element of the encephalomyocarditis virus (EMCV) was employed. However, this element, which is highly efficient in mammalian systems, did not promote efficient internal translation of the second cistron in various insect cells lines originating from different insect species. The lack of efficient internal translation was not due to baculovirus propagation since the same phenomenon was also observed in a viral-free expression system. It seems that a component essential for efficient EMCV IRES activity is either missing or present in limiting amount in insect cells or not compatible. Nevertheless, LUC placed downstream to the IRES element, or immediately downstream to the first cistron, was expressed to a level that enabled the biotechnological application it was designed for.

Animals↗