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PubMedDate not supplied

Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells.

The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells. The hsp70 protein coprecipitated with the immunoglobulin to indicate the formation of a specific hsp70-immunoglobulin complex in vivo. Immunoblot and pulse chase studies indicated that coexpression of hsp70 increased intracellular immunoglobulin solubility. Metabolic labeling experiments revealed that hsp70 increased secreted immunoglobulin levels after several days infection as compared to infection with control baculoviruses. Pulse chase studies indicated that hsp70 increases the solubility of immunoglobulin precursors that are then processed and assembled into the complete antibody oligomer. A comparison of the action of cytosolic hsp70 chaperone to the endoplasmic reticulum chaperone BiP suggests sequential action in which hsp70 increases the solubility of preprocessed immunoglobulin, while BiP enhances the solubility of processed immunoglobulin chains.

Animals↗
PubMed1994

Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells.

Rotaviruses are triple-layered particles that contain four major capsid proteins, VP2, VP4, VP6, and VP7, and two minor proteins, VP1 and VP3. We have cloned each of the rotavirus genes coding for a major capsid protein into the baculovirus expression system and expressed each protein in insect cells. Coexpression of different combinations of the rotavirus major structural proteins resulted in the formation of stable virus-like particles (VLPs). The coexpression of VP2 and VP6 alone or with VP4 resulted in the production of VP2/6 or VP2/4/6 VLPs, which were similar to double-layered rotavirus particles. Coexpression of VP2, VP6, and VP7, with or without VP4, produced triple-layered VP2/6/7 or VP2/4/6/7 VLPs, which were similar to native infectious rotavirus particles. The VLPs maintained the structural and functional characteristics of native particles, as determined by electron microscopic examination of the particles, the presence of nonneutralizing and neutralizing epitopes on VP4 and VP7, and hemagglutination activity of the VP2/4/6/7 VLPs. The production of VP2/4/6 particles indicated that VP4 interacts with VP6. Cell binding assays performed with each of the VLPs indicated that VP4 is the viral attachment protein. Chimeric particles containing VP7 from two different G serotypes also were obtained. The ability to express individual proteins or to coexpress different subsets of proteins provides a system with which to examine the interactions of the rotavirus structural proteins, the role of individual proteins in virus morphogenesis, and the feasibility of a subunit vaccine.

Animals↗
PubMed1999

Expression and characterization of glycolipid-anchored B7-1 (CD80) from baculovirus-infected insect cells: protein transfer onto tumor cells.

Tumor cells can be modified to express immunostimulatory molecules such as B7-1 by protein transfer using purified glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1). In this study recombinant baculovirus encoding GPI-B7-1 (vBacB7-1(GPI)) was established to obtain large quantities of purified GPI-B7-1 to modify tumor cells by protein transfer. vBacB7-1(GPI)-infected insect cells showed high-level cell surface expression of GPI-B7-1 that was susceptible to PIPLC treatment. GPI-B7-1 expressed in insect cells (Bac-GPI-B7-1) mediated T cell proliferation, indicating that the GPI-B7-1 retains costimulatory activity. Moreover, Bac-GPI-B7-1 was completely solubilized in Triton X-100 at 4 degrees C compared to 22% solubilization of GPI-B7-1 expressed in CHOK1 cells, suggesting that GPI-anchored proteins expressed in insect cells may not be clustered into the detergent-insoluble fraction. SDS-PAGE analysis of Bac-GPI-B7-1 showed faster mobility (45 kDa) compared to GPI-B7-1 from CHOK1 (68 kDa) and this difference may be due to a difference in glycosylation. Cell binding assays showed that immunoaffinity-purified Bac-GPI-B7-1 retained its functional ability to bind CD28(+) cells. Moreover, when human tumor cells were incubated with this functionally active purified GPI-B7-1, an efficient transfer of B7-1 onto tumor cells was observed. These results demonstrate that GPI-B7-1 can be expressed in insect cells in a functionally active form and can be used to modify tumor cells for immunotherapeutic applications.

Animals↗
PubMed1995

Odorant-binding proteins in insects.

This paper reviews the characteristics of pheromone and odorant-binding proteins (OBP) in insects, with particular reference to Lepidoptera. They are small (15 kDa) soluble proteins, very concentrated in the lymph of chemosensory sensilla and belonging to two major classes, pheromone-binding proteins (PBP) and general odorant-binding proteins. They represent the insect equivalent of vertebrate OBP. The main unsolved question with OBP of insects and vertebrates regards their physiological role in olfactory transduction. The recent discovery of several types of OBP in the same animal species suggests that these proteins may be involved in the discrimination of odours.

Amino Acid Sequence↗
PubMed2005

Gene design of signal sequence for the effective secretion of recombinant protein using insect cell.

In order to increase the proteinic secretion quantity of production in insect cell, the chicken lysozyme signal peptide (CLSP) was altered, and the effect of mutations on secretion of human lysozyme (HLY), the bovine interferon beta-3, and the equine interferon alpha-1 were studied. A hybrid gene composed of CLSP gene and HLY gene was used for the production of recombinant HLY in insect cell (BmN4) using vacuole virus expression system. In contrast with the Leu rich signal peptide in previous reports, Val rich type, or Ile rich type decreased the secretion of HLY, and Phe rich type prevented it completely, in spite of the increase of hydrophobicity in the central core region of each signal peptide. Besides the hydrophobicity, the tertiary structure in this region also might influence the secretion of HLY in insect cells. Then, Leu-rich signal peptide was used for the secretion of the bovine interferon beta-3, or the equine interferon alpha-1, resulted in the remarkable increase of the amount of the secretion in insect cells.

Amino Acid Sequence↗
PubMed1994

The bilin-binding protein of Pieris brassicae. cDNA sequence and regulation of expression reveal distinct features of this insect pigment protein.

The bilin-binding protein (BBP) is a blue pigment protein which is abundant in the butterfly Pieris brassicae. In an attempt to clarify the physiological role of this member of the lipocalin family of proteins, its complete cDNA was cloned and expression of the BBP gene in P. brassicae was investigated. It was found that synthesis of the BBP mRNA is highly regulated during the insect's ontogenesis. In larvae after the third ecdysis as well as in pupae and adults, large amounts of the mRNA are present. Each of these stages itself displays a distinct time course of mRNA synthesis. In addition, BBP is expressed tissue specifically, with the fat body being the major source of this secretory protein in the larvae. Hence, the expression pattern of BBP in this organism is markedly different from the closely related pigment protein insecticyanin in Manduca sexta. Finally, the bacterial expression of BBP in a functional state was established as a basis for the future analysis of its ligand-binding functions by protein engineering.

Amino Acid Sequence↗
PubMed2003

Expression and purification of polyhistidine-tagged rotavirus NSP4 proteins in insect cells.

The rotavirus nonstructural NSP4 protein, a transmembrane endoplasmic reticulum-specific glycoprotein, has been described as the first viral enterotoxin. Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induces diarrhea in young mice. NSP4 has a membrane-destabilizing activity and causes an increase in intracellular calcium levels and chloride secretion by a calcium-dependent signalling pathway in eucaryotic cells. In this study, four recombinant baculoviruses were generated expressing the rotavirus NSP4 glycoprotein from the human strains Wa and Ito, the porcine strain OSU, and the simian strain SA11, which belong to two different NSP4 genotypes, A and B. The recombinant glycoproteins, expressed as polyhistidine-tagged molecules, were analyzed by Western blotting and immunoprecipitation. Newborn mice responded with diarrhea after inoculation with each of the recombinant NSP4 proteins.

Animals↗
PubMed1998

Characterization of the interactions of human papillomavirus type 16 E6 with p53 and E6-associated protein in insect and human cells.

Human papillomavirus (HPV) 16 E6 induces the degradation of the tumour suppressor protein p53 by the ubiquitin-dependent proteolysis pathway. In vitro, this process involves the formation of a trimolecular complex between E6, p53 and a cellular protein E6-associated protein (E6-AP). However, an analysis of their potential interactions in vivo has not been carried out. We have established a model for the expression and analysis of the interactions of these three proteins in insect cells, a eukaryotic system where potentially crucial modifications of the proteins will occur. In baculovirus-infected cells the degradation of p53 can occur. However, p53 is only degraded early in the infectious cycle due to a lack of ATP at later times. Consequently, substantial quantities of material can be produced in this system for further analysis. Evidence is also provided that, in vivo, E6 can interact with p53 in the absence of E6-AP and that E6-AP can interact with p53 in the absence of E6. Furthermore, analysis of the subcellular localization of the proteins using both biochemical fractionation and indirect immunofluorescence suggests that the degradation of p53 occurs in the perinuclear region of the cell.

Animals↗
PubMed1996

Expression and characterization of the multidrug resistance-associated protein in insect cells infected with a recombinant baculovirus.

The protein encoded by the multidrug resistance-associated protein (MRP) gene was examined after infection of SF21 insect cells with recombinant baculovirus containing a full-length MRP cDNA. The time course of appearance of the protein as determined by western blot analysis revealed that maximum levels occurred 2 days postinfection. The amount of MRP made in this system was somewhat variable, but levels that were about 4-fold greater than that found in HL60/ADR cells could be achieved. The protein appeared to be full-length but was present in a highly deglycosylated form. The P170 (MRP) was phosphorylated and located exclusively in membranes of infected cells. P170 (MRP) synthesized in this system was capable of carrying out the ATP-dependent transport of leukotriene C4 into isolated membrane vesicles. The results thus indicate that MRP synthesized in insect cells is functional and has properties similar to the authentic protein found overexpressed in certain multidrug-resistant isolates.

ATP-Binding Cassette Transporters↗
PubMed1994

Polydnavirus infection inhibits synthesis of an insect plasma protein, arylphorin.

The wasp Campoletis sonorensis injects a segmented, double-stranded DNA polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. After parasitization, CsPDV protects the wasp egg and larva by selectively disabling the host's cellular immune response. Other host physiological systems including growth and development are affected to the apparent benefit of the parasite. To begin the characterization of the biochemical effects and mode of action of CsPDV on host growth, the titre of a developmentally regulated insect storage protein, arylphorin, was studied. Parasitized or virus-infected insects had substantially less circulating arylphorin than control insects. Fat bodies from parasitized larvae also synthesized less arylphorin in vitro. However, Northern blots of total RNA from parasitized and non-parasitized, control insects showed that the arylphorin transcript level was unaffected by parasitization suggesting a biochemical block at the translational level. In vitro translation followed by immunoprecipitation of arylphorin indicated that the mRNA was present and translatable at equal levels in both parasitized and control insects. Injection of purified virus elicited the response observed in naturally parasitized larvae, demonstrating that the effect on arylphorin synthesis is mediated, either directly or indirectly, by polydnavirus gene product(s).

Animals↗
PubMed1985

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.

Animals↗
PubMed2006

Soluble proteins in insect chemical communication.

Our understanding of the biochemical mechanisms that mediate chemoreception in insects has been greatly improved after the discovery of olfactory and taste receptor proteins. However, the presence of soluble polypeptides in high concentration around the dendrites of sensory neurons still poses unanswered questions. More than 2 decades after their discovery and despite the wealth of structural information available, the physiological function of odorant-binding proteins is not well understood. More recently, members of a second family of soluble polypeptides, the chemosensory proteins, were also discovered in the lymph of chemosensilla. Here we review the structural properties of both classes of soluble proteins, their affinity to small ligands, and their expression in the different parts of the insect body and subcellular localisation. Finally, we discuss current ideas and models of the role of such proteins in insect chemoreception.

Amino Acid Sequence↗
PubMed1990

Identification and characterization of the gene encoding the major structural protein of insect iridescent virus type 22.

The major structural protein (MSP--apparent molecular weight 49,000) of insect iridescent virus type 22 (isolated from blackflies--Simulium spp.) was resolved from disrupted, purified virus particles by SDS-PAGE and transferred to nitrocellulose by Western blotting. The portion of the blot containing the MSP was identified and excised. Tryptic peptides, generated by digestion in situ, were purified by HPLC. Three of these peptides were sequenced and an oligonucleotide gene probe was designed using one of them. A SalI clone of IV22 DNA was identified as MSP-specific by hybridization. DNA from this and an overlapping DNA clone was sequenced and a large open reading frame was positively identified as the MSP coding sequence by comparison with the tryptic peptide sequences. The molecular weight of the predicted protein product of this gene is 51,993, comparable with the apparent weight obtained by SDS-PAGE. In infected Spodoptera frugiperda (Sf) cells MSP is synthesized from 12 hr postinfection onwards. The identification of this gene and analysis of its expression opens the way to elucidating the control of late gene expression in an insect iridescent virus.

Amino Acid Sequence↗
PubMed1998

Expression of human activin C protein in insect larvae infected with a recombinant baculovirus.

In order to generate dimeric recombinant transforming growth factor-beta (TGF-beta) proteins, expensive eucaryotic cell systems, such as CHO cells, are usually used. An alternative represents the expression of such proteins in insects using a baculovirus expression system. In this study, recombinant human activin C protein was expressed in Noctuidae larvae. On SDS-PAGE, the expressed protein has a size of about 15 kD under reducing conditions and of about 20 kD under non-reducing conditions. This suggests that activin C is expressed as a dimer and disulfide bridges can be formed. Compared with expression in eucaryotic cell culture systems, expression in insect larvae presents a rapid and low cost method, without the need for expensive tissue culture scale-ups or special equipment.

3T3 Cells↗
PubMed1994

Function of antimicrobial proteins in insects.

We have isolated and characterized various antimicrobial proteins from the haemolymph of Sarcophaga peregrina (flesh fly) larvae. Of these the sarcotoxin I family is a group of proteins mainly active against Gram-negative bacteria whereas sapecin is active mainly against Gram-positive bacteria. In addition to its function in defence, sapecin also plays a role in insect development. Recently, we identified a hendecapeptide of the sapecin homologue sapecin B that has the same antibacterial activity as the original sapecin B. Both sarcotoxin I and sapecin are inducible proteins synthesized de novo by the fat body and/or haemocytes and secreted into the haemolymph when the insect is in the acute phase response to bacterial infection. Antifungal protein (AFP) is constitutively present in the haemolymph and is active against certain fungi but not bacteria. These various antimicrobial proteins interact with microbial membranes. Sarcotoxin I interferes with membrane functions such as ATP synthesis and amino acid transport. The fungicidal activity of AFP is enhanced synergistically by sarcotoxin I, although sarcotoxin I alone has no appreciable antifungal activity. It is clear that the flesh fly has the ability to mount a potent defence response against microbial parasites by mobilizing several antimicrobial proteins.

Amino Acid Sequence↗
PubMed1992

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.

Affinity Labels↗
PubMed1991

Enhancement of insect antifreeze protein activity by antibodies.

Antifreeze proteins, produced by many cold water marine teleost fish and terrestrial arthropods (insects, spiders, etc.), inhibit ice crystal growth by a non-colligative mechanism, probably by adsorbing onto the surface of potential seed ice crystals and thereby blocking growth at preferred growth sites. In this study it is demonstrated that the activity of two insect antifreeze proteins is greatly increased by the addition of specific rabbit polyclonal antibodies to the antifreezes. A model is presented which suggests that the enhancement occurs because the antifreeze-antibody complex, being much larger than the antifreeze protein alone (a minimal 7-8-fold increase in size), blocks a larger area of the ice crystal surface and extends further above the surface, thus requiring the temperature to be further lowered before crystal growth proceeds. This idea is further supported by the finding that addition of goat anti-rabbit IgG to the antifreeze protein + anti-antifreeze protein antibody complexes further enhanced activity.

Animals↗