Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion

Summary The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells byAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9^P35AcV5-1 and Sf9^P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9^P35AcV5-1 and Sf9^P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.     

Expression of three reporter genes in four cell lines developed from <Emphasis Type="Italic">Papilio demoleus</Emphasis> Linnaeus (Lepidoptera: Papilionidae)

This paper used recombinant baculoviruses that carried three reporter genes, green fluorescent protein (GFP), β-galactosidase, and secreted alkaline phosphatase (SEAP), to infect four new cell lines from Papilio demoleus Linnaeus larvae (named RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3, and RIRI-PaDe-4). The expression levels of the three recombinant proteins were detected at 24, 48, 72, 96, 120, and 144 h after infection and compared with Sf9 and High Five cells to evaluate the characteristics of these four cell lines as host cells. The inoculation densities of the tested cell lines were 2?×?10⁴ cells/well (96-well plate) and 1?×?10⁵ cells/well (24-well plate), and adding a volume of virus stock resulted in an MOI of 5.0. The results showed that the four cell lines could be infected by recombinant baculovirus and that cell lysis occurred 96 h after infection. In the four tested cell lines, only a small number of RIRI-PaDe-1 and RIRI-PaDe-3 cells expressed recombinant GFP and showed green fluorescence. The expression was much lower than that of Sf9 and High Five. Comparing the intracellular and extracellular activity of β-galactosidase indicated that the P. demoleus cell system was more suitable for the expression of secreted proteins, and its extracellular β-galactosidase level was close to that of Sf9, but the expression level of SEAP was far lower than those of Sf9 and High Five.     

Expression of the green fluorescent protein carried by Autographa Californica multiple nucleopolyhedrovirus in insect cell lines

Summary A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID₅₀ titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to Ac-MNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401+10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401+10% FBS (37.8×10⁶ TCID₅₀/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4×10⁶ TCID₅₀/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9×10⁶ TCID₅₀/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.     

Recombinant protein expression in aDrosophila cell line: comparison with the baculovirus system

In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinantDrosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by theDrosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [³H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.     

Human Substance P Receptor Expressed in Sf9 Cells Couples with Multiple Endogenous G Proteins

Abstract

To identify the G proteins involved in the function of human substance P receptor (hSPR), the receptor was expressed in Sf9 cells using the baculovirus expression system. Maximal hSPR expression was up to 65 pmol/mg membrane protein. The following data indicated that hSPR in Sf9 membranes is coupled to endogenous G proteins: 1) binding of agonist radioligand [¹²⁵I]BHSP to the receptor was sensitive to guanine nucleotides; and 2) stimulation of the receptor increased [³⁵S]GTPγS binding. The hSPR-associated G proteins were identified by photoaffinity labeling with [α-³²P]-azidoanilido GTP ([α-³²P]AAGTP), followed by immunoprecipitation of the labeled G proteins with antibodies specific for various Gα-subunits. These experiments showed that stimulation of hSPR in Sf9 membranes activated multiple endogenous G proteins including Gα_o, Gα^q/11, and Gα. While hSPR's ability to associate with G^q/11 is well-documented, the present study provides the first evidence of hSPR's potential to activate Gα_o and Gα_s.     

The Production of a Stably Transformed Insect Cell Line Expressing An Invertebrate GABAA Receptor β-Subunit

Abstract

We have produced a stable insect cell line derived from Spodoptera frugiperda (Sf9) cells expressing a cDNA encoding a β-subunit of the Lymnaea stagnalis GABA_A receptor. The cDNA was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (IE-1) promoter. Stable cell lines were established by transformation of Sf9 cells with the expression vector pIEK1.LGβ1 together with a plasmid encoding a selectable marker which confers neomycin (G418) resistance. Following growth in the presence of G418, neomycin resistant clones were selected, amplified and analysed for the presence of functional GABA-gated chloride channels. Electrophysiological analysis of one cell line showed the presence of a picrotoxin-sensitive chloride channel not present in control Sf9 cells. These channels were also sensitive to GABA, albeit at relatively high (mM) concentrations.     

Maximizing production of estrogen receptor beta with the baculovirus expression system

Steroid hormone/nuclear receptor expression in cultured insect cell lines is routinely driven by a baculovirus vector. An advantage of the baculovirus production of these receptors is that large amounts of functional receptors are obtained for subsequent in vitro studies. Most laboratories produce nuclear receptors in Spodoptera frugiperda (Sf)9 cells. However, no one has determined whether this cell line is optimal for the production of any nuclear receptor. We compared the time course and level of estrogen receptor beta (ER beta) production from a baculovirus in two S. frugiperda cell lines, IPLB-SF21AE (Sf21) and Sf9, and two Trichloplusia ni cell lines, Tn368 and BTI-TN5b1-4 (High Five). Cells were harvested at various times (0.5-5 days) after infection. ER beta expression and activity was determined by specific [3H]estradiol (E2) binding, Western blot analysis, and estrogen response element (ERE) binding in vitro. The highest functional, bioactive ER beta expression both at the earliest time after infection and in the amount of ER beta produced/cell was with the Sf21 cell line. Baculovirus expressed ER beta-bound EREs with high affinity in a DNA sequence-dependent manner. We conclude that Sf21 cells are the best-suited cells for ER beta production.     

A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells

Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.     

Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

The baculovirus–insect cell expression system has been used to produce functional recombinant proteins. The antigen GA733 is a cell‐surface glycoprotein highly expressed on most human colorectal carcinoma cells. Conditions for the expression of GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) were optimized in the baculovirus expression system. Several variable factors were adjusted to optimize expression, including the cell line (Sf9 and High Five), multiplicity of infection (MOI) value (0.05, 0.1, 0.5, 1 and 3), post‐infection time (48, 72 and 96 h) and harvested sample (cell culture media (CM) or cell lysate (CL)). In addition, two pFastBac Dual vectors carrying the GA733‐Fc gene were constructed to express GA733‐Fc with or without an endoplasmic reticulum (ER) retention sequence KDEL and used to generate recombinant baculoviruses. Western blot showed that expression depended on the conditions used to express the recombinant proteins. The protein production level and secretion capability differed in each cell line. In Sf9 cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 96 h post‐infection at 0.1 MOI and with GA733‐FcK at 96 h post‐infection at 3 MOI, respectively. In High Five cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 48 h post‐infection at 1 MOI and with GA733‐FcK at 48 h post‐infection at 3 MOI, respectively. These results suggest that the MOI value, post‐infection time and subcellular localization affect expression, and that these conditions can be modified to optimize protein expression in the baculovirus–insect cell system.     

Expression of rice gall dwarf virus outer coat protein gene (<Emphasis Type="Italic">S8</Emphasis>) in insect cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48–72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.     

Comparative characterization of growth and recombinant protein production among three insect cell lines with four kinds of serum free media

Three insect cell lines, Sf9, Sf21 and Tn5B1-4, and four different kinds of serum free media (SFM), Sf 900 II, EX-CELL 420, EX-CELL 405 and Express Five, were used to compare the nutrient consumption, byproduct formation, production of recombinant protein and protease activity in suspension cultures. The Sf 900 II SFM was appropriate for the cell growth and protein production of the Sf9 and Sf21 cell lines. When the Tn5B1-4 cell line was grown in the Express Five SFM, the specific growth rate was 1.6 fold higher than those of either the Sf9 or Sf21 cell lines. The glucose and glutamine consumption rates per cells, were 4 and 2.3. times higher than those of the Sf9 cell line, respectively. The overall yield coefficients of the lactate and ammonium ion were 2.8 and 1.5 times higher compared to those of the Sf9 cell line, respectively. The maximum specific β-galactosidase production rate was 4.5. fold that of the Sf9 cell line, a 3 times higher protease activity per cell.     

Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100–150 mM. It was recently discovered that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI) with 9–11 predicted transmembrane spanning domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12 cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes. The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake in vitro using lipid-detergent vesicles. Published: June 7, 2004.     

High-Level Expression and Purification of a Recombinant Human α-1,3-Fucosyltransferase in Baculovirus-Infected Insect Cells

A human α-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 μg/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG–Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed α-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal α-2,3-linked sialic acid among various acceptors. The apparentK_mvalues of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.     

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.     

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling

A previous study observed bell-shaped concentration-response isotherms for activation of Gα_i3 G-protein subunits by high efficacy 5-HT_1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gα_i3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT_1A receptors in other cellular environments.Here, using an antibody capture-based [³⁵S]GTPγS binding assay for Gα_i3 activation, we investigated whether efficacious 5-HT_1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (−)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT_1A receptors.In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly ‘silent’ for Gα_i3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gα_i3 activation.These data indicate that Gα_i3 G-protein activation by 5-HT_1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.     

Heterologous expression of human Neuromedin U receptor 1 and its subsequent solubilization and purification

Human Neuromedin U receptor 1 (hNmU-R1) is a member of G protein-coupled receptor family. For structural determination of hNmU-R1, the production of hNmU-R1 in milligram amounts is a prerequisite. Here we reported two different eukaryotic expression systems, namely, Semliki Forest virus (SFV)/BHK-21 and baculovirus/Spodoptera frugiperda (Sf9) cell systems for overproduction of this receptor. In the SFV-based expression system, hNmU-R1 was produced at a level of 5 pmol receptor/mg membrane protein and the yield could be further increased to 22 pmol receptor/mg membrane protein by supplementation with 2% dimethyl sulfoxide (DMSO). Around 8 pmol receptor/mg membrane protein could be achieved in baculovirus-infected Sf9 cells. The recombinant hNmU-R1 from SFV- and baculovirus-based systems was functional, with a K_d value of [¹²⁵I] NmU-23 (rat) similar to that from transiently transfected COS-7 cells, where hNmU-R1 was first identified. With the aid of 1% n-dodecyl-β-d-maltoside (LM)/0.25% cholesteryl hemisuccinate (CHS), the yield of functional hNmU-R1 could reach 80%. The recombinant receptor from Sf9 cells was purified to homogeneity. The specific binding of the purified receptor to [¹²⁵I] NmU-23 (rat) indicated that the receptor is bioactive. This is the first report of successful solubilization and purification of hNmU-R1, and will enable functional and structural studies of the hNmU-R1.     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene 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, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex‐type N‐glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P‐vank‐1), which encodes an anti‐apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High Five^TM cells, and SfSWT‐4 cells, which can produce glycoproteins with complex‐type N‐glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N‐glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P‐vank‐1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT‐4 cells were infected with a vankyrin‐encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin‐expressing cells were combined with a vankyrin‐encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin‐encoding baculovirus vectors. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1496–1507, 2017     

Comparison of oligosaccharide processing among various insect cell lines expressing a secreted glycoprotein

Summary The processing of the N-linked oligosaccharide modifying a secreted alkaline phosphatase glycoprotein (SEAP) expressed with a recombinantAutographa californica nuclear polyhedrosis virus was evaluated in insect cell lines established fromSpodoptera frugiperda, Trichoplusia ni, andMamestra brassicae. Studies with Endoglycosidase H (Endo H), which removes high-mannose oligosaccharides, revealed that 79% of the intracellular SEAP produced in theM. brassicae-derived MB0503 cell line was Endo H resistant. The commonly usedS. frugiperda Sf21 and Sf9 cell lines produced 44 and 21% Endo H-resistant intracellular SEAP, respectively. Detection of oligosaccharide moieties with lectins, which selectively recognize terminal sugars, identified only mannose residues on SEAP expressed in the six insect cell lines. However, the oligosaccharide moiety of SEAP expressed in a Chinese hamster ovary cell line contained sialic acid. Therefore, when expressed in mammalian cells, the oligosaccharide present on SEAP is processed into complex oligosaccharide, but in insect cells it is of the high-mannose type. Studies with inhibitors of the initial oligosaccharide processing steps demonstrated that all six cell lines possessed glycosidase I/II and mannosidase I activity and that glycosylation was required for secretion.