scholarly journals Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line

1992 ◽  
Vol 284 (1) ◽  
pp. 53-59 ◽  
Author(s):  
F Fossiez ◽  
G Lemay ◽  
N Labonté ◽  
F Parmentier-Lesage ◽  
G Boileau ◽  
...  
Keyword(s):  
Insect Cell ◽  
Mammalian Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Insect Cell Line ◽  
Cell Types ◽  
Neutral Endopeptidase ◽  
Cell System ◽  
Recombinant Enzyme ◽  
Soluble Form

Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.

scholarly journals A COMPARISON OF SURVIVAL AND REPAIR OF UV-INDUCED DNA DAMAGE IN CULTURED INSECT VERSUS MAMMALIAN CELLS

Genetics ◽  
1977 ◽  
Vol 87 (3) ◽  
pp. 513-518
Author(s):  
Thomas M Koval ◽  
Ronald W Hart ◽  
Willard C Myser ◽  
Walter F Hink
Keyword(s):  
Dna Damage ◽  
Cell Line ◽  
Insect Cell ◽  
Colony Formation ◽  
Mammalian Cells ◽  
Insect Cell Line ◽  
Cell Types ◽  
Unscheduled Dna Synthesis ◽  
Mammalian Cell Line ◽  
Lethal Effects

ABSTRACT Survival and unscheduled DNA synthesis (UDS) were measured in a cultured insect cell line, TN-368, and a cultured mammalian cell line, V-79-4, following exposure to several fluences of ultraviolet light. TN-368 cells were approximately seven times more resistant to the lethal effects of UV than V-79 cells, as determined by colony formation. The amount of UDS per unit amount of DNA is about the same in both cell types 4 hr after 10-50 J/m2 UV irradiations.

scholarly journals Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

2002 ◽  
Vol 68 (9) ◽  
pp. 4583-4592 ◽  
Author(s):  
Neema Agrawal ◽  
Pawan Malhotra ◽  
Raj K. Bhatnagar
Keyword(s):  
Insect Cell ◽  
Spodoptera Litura ◽  
Expression System ◽  
Insect Cell Line ◽  
Baculovirus Expression System ◽  
Aminopeptidase N ◽  
Receptor Protein ◽  
Binding Studies ◽  
Toxin Binding ◽  
Cry Toxin

ABSTRACT Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.

High-level expression of recombinant immunoreactive thyroid peroxidase in the High Five insect cell line

1996 ◽  
Vol 17 (2) ◽  
pp. 165-174 ◽  
Author(s):  
F Grennan Jones ◽  
A Wolstenholme ◽  
S Fowler ◽  
S Smith ◽  
K Ziemnicka ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Insect Cell ◽  
Culture Medium ◽  
Aminolevulinic Acid ◽  
Expression System ◽  
Insect Cell Line ◽  
Thyroid Peroxidase ◽  
High Five Cells ◽  
Insect Cell Lines

ABSTRACT Expression of a major thyroid autoantigen, thyroid peroxidase (TPO) was studied using the baculovirus-insect cell expression system. Human TPO cDNA modified so as to code for the extracellular fragment of the protein was placed under the control of the strong polyhedrin promoter in baculovirus transfer vector pBlueBacIII and cotransfected with linearized AcMNPV viral DNA. Expression in two insect cell lines Spodoptera frugiperda (Sf9) and Tricoplusia ni (High Five) was investigated and levels of recombinant TPO (rTPO) monitored by RIA and SDS-PAGE followed by Western blotting. Both insect cell lines expressed rTPO, but higher levels (30 mg/l culture medium) were obtained with High Five cells. Culture medium rTPO was purified and its glycosylation and immunoreactivity analysed. Lectin-affinity blotting and treatment with glycosidases indicated that both high mannose and complex-type sugar residues were associated with the recombinant protein. Studies with an ELISA based on biotin-labelled rTPO and an immunoprecipitation assay based on 125I-labelled rTPO indicated that the rTPO and native TPO showed similar reactivity to TPO autoantibodies (r=0·96, P<0·001, n=50 and r=0·99, P<0·001, n=80 respectively). In addition, rTPO expressed in High Five cells showed enzyme activity comparable with that of native TPO when the heme biosynthesis precursor δ-aminolevulinic acid was included in the culture medium. Overall, our studies indicate that the High Five insect cell line provides a useful system for the expression of relatively high levels of rTPO which should be suitable for structural analysis of TPO and TPO—TPO autoantibody complexes.

The mRNA encoding TAFI is alternatively spliced in different cell types and produces intracellular forms of the protein lacking TAFIa activity

2013 ◽  
Vol 109 (06) ◽  
pp. 1033-1044 ◽  
Author(s):  
Joellen H. H. Lin ◽  
Dragana Novakovic ◽  
Christina M. Rizzo ◽  
Branislava Zagorac ◽  
Mathieu Garand ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Mammalian Cells ◽  
Mononuclear Cells ◽  
Recombinant Expression ◽  
Cell Types ◽  
Coagulation Cascade ◽  
Peptidase Activity ◽  
Peripheral Blood Mononuclear ◽  
Mrna Variant ◽  
Alternatively Spliced

SummaryTAFI (thrombin-activatable fibrinolysis inhibitor) is a pro-carboxypeptidase, encoded by the CPB2 gene in humans that links the coagulation cascade to fibrinolysis and inflammation. The liver is the main source for plasma TAFI, and TAFI expression has been documented in platelets and monocyte-derived macrophages. A recent study reported an alternatively spliced CPB2 mRNA variant lacking exon 7 (Δ7) in HepG2 cells and liver. Another study identified a CPB2 mRNA variant lacking exon 7 and a 52 bp deletion in exon 11 (Δ7+11) in human hippocampus. We have examined alternative splicing of CPB2 mRNA in various cell types by RT-PCR and have assessed the functional properties of TAFI variants encoded by these transcripts by recombinant expression in mammalian cells. We identified the Δ7 exon skipping event in liver, Dami megakaryoblasts, THP-1-derived macrophages, peripheral blood mononuclear cells, platelets, testis, cerebellum, and SH-SY5Y neuroblastoma cells. The Δ11 alternative splicing event was notably absent in liver cells. We also detected a novel exon Δ7+8 skipping event in liver and megakaryocytes. Of note, we detected non-alternatively spliced CPB2 transcripts in brain tissues, suggesting the expression of full-length TAFI in brain. Experiments using cultured mammalian cells transfected with wild-type CPB2-, Δ7-, Δ7+11 -, and_Δ11 -cDNA revealed that alternatively spliced TAFI is stored inside the cells, cannot be activated by thrombin-thrombomodulin, and does not have TAFIa activity. The alternative splicing events clearly do not give rise to a secreted protein with basic carboxy-peptidase activity, but the intracellular forms may possess novel functions related to intracellular proteolysis.

scholarly journals Differential Incorporation of Cholesterol by Sindbis Virus Grown in Mammalian or Insect Cells

2009 ◽  
Vol 83 (18) ◽  
pp. 9113-9121 ◽  
Author(s):  
Amanda Hafer ◽  
Rebecca Whittlesey ◽  
Dennis T. Brown ◽  
Raquel Hernandez
Keyword(s):  
Cell Membrane ◽  
Cell Lines ◽  
Insect Cell ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Virus Assembly ◽  
Critical Role ◽  
Insect Cell Line

ABSTRACT Cholesterol has been shown to be essential for the fusion of alphaviruses with artificial membranes (liposomes). Cholesterol has also been implicated as playing an essential and critical role in the processes of entry and egress of alphaviruses in living cells. Paradoxically, insects, the alternate host for alphaviruses, are cholesterol auxotrophs and contain very low levels of this sterol. To further evaluate the role of cholesterol in the life cycle of alphaviruses, the cholesterol levels of the alphavirus Sindbis produced from three different mosquito (Aedes albopictus) cell lines; one other insect cell line, Sf21 from Spodoptera frugiperda; and BHK (mammalian) cells were measured. Sindbis virus was grown in insect cells under normal culture conditions and in cells depleted of cholesterol by growth in serum delipidated by using Cab-O-sil, medium treated with methyl-β-cyclodextrin, or serum-free medium. The levels of cholesterol incorporated into the membranes of the cells and into the virus produced from these cells were determined. Virus produced from these treated and untreated cells was compared to virus grown in BHK cells under standard conditions. The ability of insect cells to produce Sindbis virus after delipidation was found to be highly cell specific and not dependent on the level of cholesterol in the cell membrane. A very low level of cholesterol was required for the generation of wild-type levels of infectious Sindbis virus from delipidated cells. The data show that one role of the virus membrane is structural, providing the stability required for infectivity that may not be provided by the delipidated membranes in some cells. These data show that the amount of cholesterol in the host cell membrane in and of itself has no effect on the process of virus assembly or on the ability of virus to infect cells. Rather, these data suggest that the cholesterol dependence reported for infectivity and assembly of Sindbis virus is a reflection of differences in the insect cell lines used and the methods of delipidation.

scholarly journals GPCR-Based Bioactive Peptide Screening Using Phage-Displayed Peptides and an Insect Cell System for Insecticide Discovery

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 583
Author(s):  
Man-Yeon Choi ◽  
Robert K. Vander Meer
Keyword(s):  
Pest Management ◽  
Insect Cell ◽  
Bioactive Peptides ◽  
Insect Pest ◽  
Expression System ◽  
Management Strategies ◽  
Screening Method ◽  
Risk Process ◽  
Cell System ◽  
Bioactive Peptide

The discovery of new insecticides improves integrated pest management (IPM), but is usually a long high-risk process with a low probability of success. For over two decades, insect neuropeptides (NPs) and their G-protein coupled receptors (GPCRs) have been considered as biological targets for insect pest control, because they are involved in almost all physiological processes associated with insect life stages. A key roadblock to success has been the question of how large volume chemical libraries can be efficiently screened for active compounds. New genomic and proteomic tools have advanced and facilitated the development of new approaches to insecticide discovery. In this study, we report a novel GPCR-based screening technology that uses millions of short peptides randomly generated by bacteriophages, and a method using an insect Sf9 cell expression system. The fire ant is a good model system, since bioactive peptides have been identified for a specific GPCR. The novel small peptides could interfere with the target GPCR-ligand functions. Therefore, we refer to this new mechanism as “receptor interference” (RECEPTORi). The GPCR-based bioactive peptide screening method offers multiple advantages. Libraries of phage-displayed peptides (~109 peptides) are inexpensive. An insect cell-based screening system rapidly leads to target specific GPCR agonists or antagonists in weeks. Delivery of bioactive peptides to target pests can be flexible, such as topical, ingestion, and plant-incorporated protectants. A variety of GPCR targets are available, thus minimizing the development of potential insecticide resistance. This report provides the first proof-of-concept for the development of novel arthropod pest management strategies using neuropeptides, and GPCRs.

Recovery of infective juveniles of the entomopathogenic nematode Steinernema carpocapsae via factors produced by insect cells and symbiotic bacteria

Nematology ◽  
2009 ◽  
Vol 11 (4) ◽  
pp. 611-618 ◽  
Author(s):  
Shingo Kikuta ◽  
Takashi Kiuchi ◽  
Fugaku Aoki ◽  
Masao Nagata
Keyword(s):  
Cell Lines ◽  
Insect Cell ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Entomopathogenic Nematode ◽  
Insect Cell Line ◽  
Fruit Fly ◽  
Steinernema Carpocapsae ◽  
Symbiotic Bacteria ◽  
Insect Cell Lines

Abstract Entomopathogenic nematodes, Steinernema carpocapsae, show 'recovery' from the dauer form as infective juveniles (IJ) up to fourth-stage juveniles when host invasion occurs. This recovery also occurs within an insect cell line culturing system. Here we addressed the factor(s) that induce recovery. When IJ were exposed to cell medium obtained from the cultivation of Sf9 cell lines derived from armyworms (Spodoptera frugiperda), approximately 50% of IJ recovered after 4 h. By 16 h, 90% of the IJ had undergone recovery. Other insect cell lines such as silkworm (Bombyx mori)-derived BmN cells and fruit fly (Drosophila melanogaster)-derived S2 cells also secreted the recovery inducing factor(s). By contrast, mammalian cells (NIH/3T3 and HeLa) had no effect on nematode recovery. Our data also suggest that symbiotic bacteria are involved in IJ recovery; axenic IJ did not recover in the cell-cultured medium. When symbiotic bacteria isolated from IJ were propagated within the cell-cultured medium, the supernatant gained recovery-inducing activity against axenic IJ. From these results, we conclude that IJ recovery in S. carpocapsae is induced by multiple factor(s) secreted from insect cells and symbiont bacteria.

Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system

2008 ◽  
Vol 389 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Stephan Frey ◽  
Martin Haslbeck ◽  
Otmar Hainzl ◽  
Johannes Buchner
Keyword(s):  
Mammalian Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Basic Research ◽  
Medical Diagnostics ◽  
Free Expression ◽  
Translation System ◽  
Functional Antibodies ◽  
The Stability

Abstract Antibodies are an important component of the immune system of higher eukaryotes. Furthermore, they are effective tools in basic research, medical diagnostics and therapy. Recombinant expression of these heterotetrameric, disulfide-bridged proteins is usually performed in mammalian cells. Here, we describe the cell-free expression of a mouse monoclonal antibody, MAK33, in a coupled transcription/translation system, based on an Escherichia coli lysate. Both the heavy and the light chain can be produced efficiently in this setup. However, they fail to form functional antibodies. With a view to overcome folding and oxidation defects, we supplemented the system with the oxidoreductases PDI (protein disulfide isomerase) and DsbC and the ER-specific chaperones Grp94 and BiP; furthermore, we optimized the redox conditions. We found that functional antibodies can only be obtained in the presence of an oxidoreductase. In contrast, the addition of Grp94 and/or BiP had no influence on the productive folding reaction. The comparison of the antibody expressed in vitro with MAK33 expressed in cell culture showed that the in vitro expressed antibody is correctly assembled, disulfide-bridged and shows identical antigen affinity. The stability of the in vitro expressed non-glycosylated IgG is comparable to that of the authentic antibody.

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