Insect Cell Line Sf9 Research Articles

Propagation ofSpodoptera frugiperda cells (Sf9) and production of recombinant proteins with the baculovirus expression system using improved spinner flasks with membrane aeration

It has been shown that the growth of Spodoptera frugiperda cells is significantly reduced or ceased under oxygen limiting culture conditions. This paper describes the use of a new membrane-aerated spinner flask which was compared to conventional surface-aerated spinner flasks with regard to growth of the insect cell line Sf9 and recombinant protein production after infection with baculovirus. Using a commercially available serum-free culture medium Sf9 cells reached highest cell densities (3×106 ml−1) in the membrane-aerated spinner flask. Production of recombinant protein was also influenced by the oxygen supply. In the membrane-aerated spinner flask and in a surface-aerated spinner flask with reduced filling volume more than 20000 U ml−1 of a recombinant interleukin-2 variant were accumulated whereas only 100 U ml−1 were produced in a surface-aerated spinner flask with insufficient oxygen supply. Sufficient oxygenation appears to be essential for proliferation of Sf9 cells as well as recombinant protein production after infection with baculovirus. Membrane oxygenation allows sufficient oxygen supply at high cell density and an at least 2.5 fold higher filling volume per spinner unit.

Expression of the Human Interleukin–2 Receptor Gamma Chain in Insect Cells Using a Baculovirus Expression Vector

The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates, prepared from insect cells infected with the recombinant virus. At 40 h post infection the corresponding protein was detected as two major bands with apparent molecular weights of 50-60 kDa using a rabbit anti-human IL-2R gamma-receptor specific antiserum. Metabolic labelling with [35S]-methionine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma- protein could also be determined on the surface of infected insect cells by flow cytometer analysis.

Inactivation in ShakerB K+ channels: a test for the number of inactivating particles on each channel

Fast inactivation in ShakerB K channels results from pore-block caused by "ball peptides" attached to the inner part of each K channel. We have examined the question of how many functional inactivating balls are on each channel and how this number affects inactivation and recovery from inactivation. To that purpose we expressed ShakerB in the insect cell line Sf9 and gradually removed inactivation by perfusing the cell interior with the hydrolytic enzyme papain under whole cell patch clamp. Inactivation slows down as the balls are removed by an amount consistent with the presence of four balls on each channel. Recovery from inactivation has the same time course early and late in papain action; it does not depend on the number of balls remaining on the channel, consistent with the idea that reinactivation is not significant during recovery from inactivation. Our conclusion is that ShakerB has four ball peptides, each capable of causing inactivation. Statistically, the balls are identical and independent. The stability of N-type inactivation by the remaining balls is not appreciably affected by removing some of the balls from a channel.

The relation between ion permeation and recovery from inactivation of ShakerB K+ channels

We have studied the relation between permeation and recovery from N-type or ball-and-chain inactivation of ShakerB K channels. The channels were expressed in the insect cell line Sf9, by infection with a recombinant baculovirus, and studied under whole cell patch clamp. Recovery from inactivation occurs in two phases. The faster of the two lasts for approximately 200 ms and is followed by a slow phase that may require seconds for completion. The fast phase is enhanced by both permeant ions (K+, Rb+) and by the blocking ion Cs+, whereas the impermeant ions (Na+, Tris+, choline+) are ineffective. The relative potencies are K+ > Rb+ > Cs+ > NH4+ >> Na+ approximately choline+ approximately Tris+. Ion permeation through the channels is not essential for recovery. The results suggest that cations influence the fast phase of recovery by binding in a site with an electrical distance greater than 0.5. Recovery from fast inactivation is voltage-dependent. With Na+, choline+, or Tris+ outside, about 15% of the channels recover in the fast phase (-80 mV), and the other 85% apparently enter a second inactivated state from which recovery is very slow. Recovery in this phase is not influenced by external ions, but is speeded by hyperpolarization.

Susceptibility of the Sf9 Insect Cell Line to Infection with Adventitious Viruses

Abstract. Sf9, the insect cell line commonly used for gene expression by recombinant baculovirus (BV), can be infected by St. Louis encephalitis (SLE) virus, a flavivirus, resulting in a persistent, productive, and cytopathic infection, while retaining the ability to be infected with a recombinant baculovirus (rBV). We now demonstrate using double immunofluorescence that single cells are dually infected with SLE virus and rBV. Fourteen additional viruses including additional flaviviruses, other arbovirus classes, vesicular stomatitis virus (VSV), and herpes simplex virus, type 1 (HSV-1) failed to produce a cytopathic effect (CPE) in Sf9 cells. Plaque assays indicated infectious virus was present for several weeks post-inoculation for Yellow fever (YF), Dengue types 1 and 2 (DEN-1 and DEN-2), Gumbo limbo (GL), Eastern equine encephalomyelitis virus (EEE), Western equine encephalomyelitis virus (WEE), HSV-1, and VSV viruses. For HSV-1, GL, EEE, WEE and VSV, but not for YF, DEN-1 or DEN-2 viruses, this could be attributed solely to survival in the Sf9 cell culture media. Of the 14 viruses tested, only HSV-1 could be detected after 2 weeks in serum-free media. The data indicate that several viruses which are pathogenic for humans are stable for long periods of time at 27°C in the serum-containing media used for cultivation of Sf9 cells. YF, DEN-1 and DEN-2 viruses may replicate in Sf9 cells at extremely low levels. This suggests that adventitious agents which do not produce obvious CPE or interfere with rBV infection or recombinant protein expression could contaminate Sf9 cell cultures or media.

Overexpression of Human DNA Topoisomerase I in Insect Cells Using a Baculovirus Vector

The 3645-bp human DNA topoisomerase I cDNA isolated by D′Arpa et al. (Proc. Natl. Acad. Sci. USA 85, 1988, 2543-2547) was integrated into the Autographa californica multiple nuclear polyhedrosis virus genome. The recombinant protein was expressed by infecting the SF9 insect cell line with this baculovirus and resulted in a 100-fold overexpression of human DNA topoisomerase I compared to the level found in human cell lines. This 100-kDa recombinant protein has the same electrophoretic mobility as the human DNA topoisomerase I from HeLa cells and is recognized by topoisomerase I-specific monoclonal antibody. The recombinant DNA topoisomerase I was isolated and purified to homogeneity with a two-step fractionation protocol and has a specific activity of 2 × 106 U/mg. Enzymatic properties such as stimulation by magnesium and inhibition by camptothecin resemble properties of the enzyme purified from human cell lines.

Glycosylation of recombinant prorenin in insect cells: the insect cell line Sf9 does not express the mannose 6-phosphate recognition signal.

Sf9 cells infected with a recombinant baculovirus containing the gene for human prorenin were cultured in the presence of [3H]mannose. In vivo labeled prorenin was isolated by immunoprecipitation from the culture medium and digested with Pronase. The oligosaccharide structures on the resulting glycopeptides were analyzed by a combination of lectin, ion-exchange, paper, and high-pressure liquid chromatography. Of the N-linked oligosaccharides isolated from the Sf9-produced prorenin, 98% were of a truncated (trimannosyl) high-mannose type, approximately two-thirds of which contained a fucose residue linked to the reducing N-acetylglucosamine. The remaining 2% constituted a mixture of high-mannose-type structures containing six, seven, or eight mannose residues; none of these structures were core-fucosylated. None of the oligosaccharide structures recovered from recombinant prorenin synthesized by Sf9 cells were phosphorylated or contained any other form of charge. Furthermore, assays for UDP-GlcNAc-lysosomal-enzyme N-acetylglucosamine phosphotransferase demonstrated no activity above background in lysates prepared from Sf9 cells. Blotting of Sf9 cell lysates with an 125I-labeled, soluble form of the cation-independent mannose 6-phosphate receptor failed to detect any proteins carrying the mannose 6-phosphate recognition signal. Taken together, the data suggest that Sf9 cells do not synthesize high-mannose-type oligosaccharides containing mannose 6-phosphate, and consequently it appears unlikely that these cells utilize the mannose 6-phosphate receptor mediated pathway for targeting of lysosomal enzymes.

Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells.

Deciphering the roles of the individual subunits of the heteromeric Na,K-ATPase in the structure, function, and assembly of this enzyme has been complicated because most expression systems have endogenous levels of Na,K-ATPase activity. This problem has become even more significant in light of the recent identification of multiple isoforms for both the alpha and beta subunits. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, affords two distinct advantages for the expression of the Na,K-ATPase; some insect cells have little or no levels of Na,K-ATPase, and baculovirus-infected cells produce extremely high levels of foreign protein. We have made two separate recombinant baculoviruses containing the rodent alpha 1 or beta 1 cDNAs and used them to infect the insect cell line Sf-9. The infected Sf-9 cells produce Na,K-ATPase subunit protein on the order of 5-10 micrograms of protein/ml of cultured cells. The rodent alpha 1 polypeptide produced in the Sf-9 cells is indistinguishable electrophoretically and antigenically from the native subunit. The expressed beta 1 subunit is also antigenically identical but has a higher electrophoretic mobility due to differential glycosylation by the infected Sf-9 cell. In contrast to other systems, when expressed alone, each individual Na,K-ATPase subunit is targeted to the infected Sf-9 plasma membrane. In contrast, when infected with a virus that induces the heavy chain of murine IgG, the infected Sf-9 cell retains the polypeptide in the endoplasmic reticulum. However, when both IgG light and heavy chains are expressed, the polypeptides are properly processed and secreted. When the Na,K-ATPase alpha 1 and beta 1 polypeptides are simultaneously expressed, they form detergent-resistant complexes that are functional. Ouabain-sensitive ATPase activity on the order of 5 mumol Pi/mg/h in infected Sf-9 membranes was dependent on the expression of both the alpha 1 and beta 1 subunits. Sodium-dependent phosphorylated intermediates were detected that were potassium- and ouabain-sensitive. No increase in ouabain-sensitive activity or phosphorylated intermediates was detected when either subunit was expressed alone. The alpha 1 beta 1-coinfected cells were also able to transport ions, as detected in 86Rb uptake experiments. Thus, the recombinant Na,K-ATPase expressed in insect cells is biologically active and is suitable for structural and functional analysis.

Derivation of a monoclonal anti-murine IL-4 antibody specific for an epitope expressed on denatured molecules

Monoclonal antibodies to interleukin 4 (IL-4) were generated by immunization with recombinant IL-4 and screening by binding to IL-4 adsorbed to plastic surfaces. Three antibodies were obtained that scored well in this assay and one, 13E1, was studied in detail. It was very effective in detecting IL-4 by Western blotting whereas a neutralizing anti-IL-4 antibody, 11B11, was 50–100-fold less sensitive as a detecting agent. Sequential immunoprecipitation and biosynthetic labelling studies indicated that the 11B11 and 13E1 epitopes are largely expressed on different forms of IL-4. 13E1 was able to detect cytosolic IL-4 both by immunohistochemical and flow cytometric analysis of fixed cells. This was routinely successful in an insect cell line (Sf9) expressing large amounts of IL-4 as a result of infection with a recombinant ‘IL-4 baculovirus’. Although stimulated D10 cells could also be shown to express IL-4 in their cytosol, positive results were not obtained in all such studies and we have failed to detect IL-4 production by normal T cells using this method. This antibody may have substantial value in detecting IL-4 by Western blots and as a tool to analyze the biosynthesis of IL-4. With suitable improvement in sensitivity, it also may prove valuable in the detection of IL-4 in the cytosol of individual cells.

The Sf9 cell line as a model for studying insect octopamine-receptors

Adenylate cyclase-coupled octopamine (OA) receptors were demonstrated in the Sf9 insect cell line derived from Spodoptera frugiperda. This receptor-enzyme complex is membrane-associated, has a requirement for GTP and is forskolin-sensitive. In intact Sf9 cells, 100 μM OA produces a 3-fold increase in cyclic AMP (cAMP) levels. These cells do not exhibit adenylate cyclase sensitive to dopamine or serotonin. This binding site shows a high affinity for OA ( K a = 3.5 μM), the formamidine, N-demethylchlordimeform, DCDM ( K a = 0.3 μM) and the imidazoline, 2,3-xylylaminomethyl-2′-imidazoline, XAMI ( K a = 0.7 μM) with a reduced affinity for α 2-selective agonists such as clonidine and UK-14304. Of 24 putative antagonists tested in this system, ergotamine, phentolamine, (+)mianserin and RX821002A were the most effective. Other antagonists such as yohimbine, propranolol and (+)butaclamol were relatively ineffective. These responses differ considerably from the pharmacological profile for the cockroach nerve cord octopamine-sensitive adenylate cyclase (OSAC) but are similar to previous results with the lepidopteran, Manduca sexta. There appears to be a closer pharmacological relationship of the Sf9 and cockroach OA receptor to the vertebrate imidazoline-preferring receptor (IPR) than the α 2-adrenergic receptor. Studies on receptor dynamics indicated that the Sf9 OSAC undergoes agonist-induced desensitization. Therefore, based on these combined data, we propose the Sf9 cell line as a convenient model system for studying the pharmacology and dynamics of lepidopteran adenylate cyclase-coupled OA receptors.

Recombinant porin of Haemophilus influenzae type b.

A protein of approximately 40 kDa in the outer membrane of Haemophilus influenzae type b (Hib) behaves as a porin and permits transmembrane diffusion of low-molecular-weight solutes. The gene for Hib porin was cloned from an M13 library of chromosomal DNA of Hib strain ATCC9795. The gene was subcloned into a new transfer vector as a prerequisite for its use in the baculovirus expression vector system. Pure recombinant virus (AcPORIN) was isolated. On infection of a cultivated insect cell line Sf9 with AcPORIN, a novel protein was detected in cell lysates, and this novel protein reacted with an anti-Hib porin monoclonal antibody. The purified recombinant Hib porin was tested for its pore-forming properties in a synthetic black lipid membrane. The biophysical activity of purified recombinant Hib porin was identical to porin isolated from the bacterial outer membrane.

Expression of a pheromone‐binding protein in insect cells using a baculovirus vector

A cDNA encoding a pheromone-binding protein from the male silkmoth Antheraea pernyi has been integrated into the genome of the Autographa californica multiple nuclear polyhydrosis virus such that the transcription was under the control of the strong polyhedrin promoter. Recombinant pheromone-binding protein was expressed in a baculovirus-infected insect cell line (Sf9) and secreted from the cells into the culture medium. Using a two-step protocol, recombinant pheromone-binding protein has been isolated and purified to homogeneity. Pheromone binding of recombinant protein has been demonstrated using a tritiated analog of (E,Z)-6,11-hexadecadienyl acetate.

Localization of the cystic fibrosis gene product in a transfected Sf9 insect cell line

The cystic fibrosis gene product (CFTR) is thought to be either directly or indirectly involved with Cl- conductance in epithelial cells in which it is produced. Utilizing the Baculovirus expression vector system, CFTR has been expressed in Sf9 insect cells which in turn display a regulated Cl- conductance employing the same techniques as those used to demonstrate Cl- conductance in epithelial cells. This was not observed in mock-infected Bgalactosidase producing or non-infected Sf9 cells.Two cell lines, one infected with CFTR expressing recombinant baculovirus and the other infected with B-galactosidase expressing recombinant baculovirus were fixed in 2% paraformaldehyde and 0.001% glutaraldehyde in 0.1 M HEPES buffer, PH7.2 for 2 hours. They were then rinsed several times with buffer, infiltrated with 2% agarose and infused with 2.3 M sucrose for at least 24 hours. The samples were then frozen by rapid immersion in liquid nitrogen cooled FREON 22 and ultrathin cryosections cut with a cryoultramicrotome and immunogold labelled with purified monclonal antibodies against CFTR (1) using the method described by Griffiths.

Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9

The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with [3H]glucosamine and infected with a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Gal beta 1-3GalNAc constituting 17 and 66% of the total number of structures, respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked alpha 1-6 to the asparagine-linked N-acetylglucosamine.

Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection.

Pluronic F-68 is a widely used protective agent in sparged animal cell bioreactors. In this study, the attachment-independent Spodoptera frugiperda Sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. First, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. Supplementing the medium with 0.2% (w/v) Pluronic F-68, however, fully protected the cells. Next, cell growth was compared in two airlift bioreactors with similar geometry but different sparger design; one of these bioreactors consisted of a thin membrane distributor, while the other consisted of a porous stainless steel distributor. The flow rates and bubble sizes were comparable in the two bioreactors. Supplementing the medium with 0.2% (w/v) Pluronic F-68 provided full protection to cells growing in the bioreactor with the membrane distributor but provided essentially no protection in the bioreactor with the stainless steel distributor. These results strongly suggest that cell damage can occur in the vicinity of the gas distributor. In addition, these results demonstrate that bubble size and gas flow rate are not the only important considerations of cell damage in sparged bioreactors. A model of cell death in sparged bioreactors is presented.

Structure of O‐glycosidically linked oligosaccharides synthesized by the insect cell line Sf9

The O-glycosidically linked oligosaccharides on the pseudorabies virus (PRV) glycoprotein gp50 synthesized by three different cell lines were studied. The intact membrane protein (gp50) was expressed in Vero cells and in the insect cell line Sf9. In addition, a truncated, secreted form lacking the transmembrane and cytoplasmic domains (gp50T), was expressed in CHO and Sf9 cells. The protein, both in intact and truncated form, synthesized by the two mammalian cells contained only the disaccharide Gal beta 1-3GalNAc, either unsubstituted or substituted with one or two sialic acid residues. By contrast, the major O-linked structure on gp50 and gp50T synthesized by Sf9 cells was the monosaccharide GalNAc. The Sf9 cells also linked lower amounts of Gal beta 1-3GalNAc to gp50 (12%) and gp50T (26%). None of the structures synthesized by Sf9 cells contained sialic acid. Measurements of the two relevant glycosyltransferases revealed that while all three cell lines contain comparable levels of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity, there is a greater variation in the levels of UDP-Gal:N-acetylgalactosamine, beta 1-3 galactosyltransferase, with the Sf9 cells containing the lowest level.

Structural features of nonionic polyglycol polymer molecules responsible for the protective effect in sparged animal cell bioreactors.

The nonionic surfactant Pluronic F-68 polyol is commonly used to protect cultured animal cells from the detrimental effects of sparging. In this study we investigated the structural features of the Pluronic F-68 molecule responsible for this protective behavior. Poly(oxyethylene)-poly(oxypropylene) block copolymer polyols of various molecular weights and percentages of hydrophobe (poly(oxypropylene], including both Pluronic and reverse Pluronic polyols, were considered. The potential toxicity of these agents was examined in the absence of sparging (i.e., in spinner flasks) by using the attachment-independent Sf9 insect cell line as a model system. Each polyol resulted in one of three distinct types of behavior in these spinner flask experiments: (1) cells lysed at an exponential rate, (2) inhibition of cell growth (i.e., no net cell growth), or (3) uninhibited cell growth. It was then shown that all of the Pluronic and reverse Pluronic polyols that did not inhibit cell growth provided protection from sparging in the bioreactors used in this study; thus, finding a polyol that protected cells was synonymous with finding one that did not inhibit cell growth. The ability of these polyols to protect animal cells in sparged bioreactors was found to correlate well with the hydrophilic-lipophilic balance (HLB). Those polyols with the largest HLB values were found to be protective agents. These poly(oxyethylene)-poly(oxypropylene) polyols were also shown to be more effective protective agents than pure poly(oxyethylene); thus, the presence of the hydrophobe (poly(oxypropylene] is important in their ability to serve as protective agents.