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PubMed1979

Purification and reverse transcription of the messenger RNA coding for the insect protein, calliphorin, isolated from larvae of the blowfly, Calliphora vicina R.-D.

Starting from poly(A)-containing RNA prepared from the fat body of larvae of the blowfly Calliphora vicina, we have purified an mRNA coding for the protein calliphorin, which is a major blowfly protein accounting for approximately 9% of the total poly(A)-containing mRNA activity in the fat body of 5-days-old larvae, as demonstrated by translation in vitro. The mRNA for this protein was purified by sucrose gradient centrifugation under denaturing conditions and identified by cell-free translation. The peak fraction of the gradient shows three bands of about 20 S after formamide/acrylamide gel electrophoresis. Using reverse transcriptase and Calliphora mRNA isolated directly from the acrylamide gel electrophoresis or from sucrose gradients, we synthesized a cDNA probe. This cDNA hybridizes with the template, showing a major kinetic component with a molecular weight of 2.8 x 10(6), fitting well with the three bands observed in the electrophoresis. Hybridization of the cDNA with total sonicated Calliphora DNA shows annealing only at very high c0t values, indicating that Calliphora mRNA is transcribed from the unique portion of the fly genome.

Adipose Tissue↗

PubMed1966

Properties of bursicon: an insect protein hormone that controls cuticular tanning.

On gel filtration the retention volume of bursicon indicates a molecular weight of about 40,000. On disc electrophoresis bursicon migrates toward the anode and appears at an R(F) (relative to the marker dye) of 0.3 to 0.4. The properties of fractions with bursicon activity in blood, brain, and ganglion of a fly (Sarcophaga bullata), and in blood, ganglion, and corpora cardiaca of a roach (Periplaneta americana) are similar, but not identical. Bursicon in the blood is more heat labile, and the activity in brain and corpora cardiaca shows two peaks in electrophoresis, instead of one as in the other fractions.

Animals↗

PubMed1995

Lipopolysaccharide-stimulated exocytosis of nonself recognition protein from insect hemocytes depend on protein tyrosine phosphorylation.

Insect hemocytes (blood cells) synthesize the major nonself recognition protein (47 kDa) during 3rd instar larvae (V.J. Marmaras, S. Tsakas, Dev. Biol. 129, 294-303 (1988)). In this study we show the presence of the 47 kDa protein in plasmatocytes (main hemocyte type) and prohemocytes. In plasmatocytes this protein appears to be localized both in vesicles and in the cell surface. The cell surface-associated 47 kDa protein was released from membrane fraction by 1 M NaCl, indicating that it is not tightly bound. Bacterial lipopolysaccharide (LPS) can function on isolated hemocytes from Ceratitis capitata larvae, inducing their spreading and degranulation. During degranulation (exocytosis) the plasmatocytes release the 47 kDa protein, among others. This protein could not be normally traced in serum, nor is it released by basal secretion. The secretion of the 47 kDa protein was found to be LPS-dependent, whereas its presence on plasmatocyte surface is LPS independent. LPS-stimulated exocytosis of the 47 kDa protein appears to be dependent on protein tyrosine phosphorylation. We have now demonstrated that LPS increases tyrosine phosphorylation of 19 and 22 kDa polypeptides in C. capitata hemocytes. Inhibition of the LPS-induced tyrosine phosphorylation mediated by tyrosine kinase inhibitor, genistein, was accompanied by the inhibition of the secretion of the 47 kDa protein. These results support the hypothesis that tyrosine protein phosphorylation is a signal reaction in hemocytes after LPS exposure. These LPS responses of insect plasmatocytes show strong similarities to mammalian macrophages (S. Weinstein et al., J. Immunol. 151, 3829-3838 (1993)). In a model we propose that the LPS-independent cell surface-associated 47 kDa protein is responsible for the phagocytosis and for the formation of nodules and capsules, whereas the LPS-dependent secreting counterpart is responsible for the extracellular killing of bacteria.

Animals↗

PubMedDate not supplied

A comparison of the composition of silk proteins produced by spiders and insects.

Proteins that are highly expressed and composed of amino acids that are costly to synthesize are likely to place a greater drain on an organism's energy resources than proteins that are composed of ingested amino acids or ones that are metabolically simple to produce. Silks are highly expressed proteins produced by all spiders and many insects. We compared the metabolic costs of silks spun by arthropods by calculating the amount of ATP required to produce their component amino acids. Although a definitive conclusion requires detailed information on the dietary pools of amino acids available to arthropods, on the basis of the central metabolic pathways, silks spun by herbivorous, Lepidoptera larvae require significantly less ATP to synthesize than the dragline silks spun by predatory spiders. While not enough data are available to draw a statistically based conclusion, comparison of homologous silks across ancestral and derived taxa of the Araneoidea seems to suggest an evolutionary trend towards reduced silk costs. However, comparison of the synthetic costs of dragline silks across all araneomorph spiders suggests a complicated evolutionary pattern that cannot be attributed to phylogenetic position alone. We propose that the diverse silk-producing systems of the araneoid spiders (including three types of protein glues and three types of silk fibroin), evolved through intra-organ competition and that taxon-specific differences in the composition of silks drawn from homologous glands may reflect limited or fluctuating amino acid availability. The different functional properties of spider silks may be a secondary result of selection acting on different polypeptide templates.

Amino Acids↗

PubMed1991

Insect antibacterial proteins: not just for insects and against bacteria.

In response to a bacterial infection, insects launch an array of countermeasures. Among these are the antibacterial proteins, which effectively lyse bacteria or are bacteriostatic. These proteins were generally assumed to be restricted to insects, yet recent information has shown some homologous counterparts in vertebrates, including humans. Recent data have revealed that at least some of these proteins can also act against eukaryotic cells, including human infectious parasites. The latter activities have opened up new possibilities for disease control.

Animals↗

PubMed1989

Use of a four-parameter logistic model to evaluate the quality of the protein from three insect species when fed to rats.

The quality of three insect protein sources [Mormon cricket meal (MCM), house cricket meal (HCM) and Eastern tent caterpillar meal (TCM)] was evaluated relative to that of lactalbumin (LA) and soy protein (SP) by using both amino acid analysis and a rat bioassay. The amino acid pattern of the three insect meals indicated that methionine should be the first limiting amino acid for growing rats. In the rat bioassay, weanling Sprague-Dawley rats were fed graded levels of the five proteins in purified diets and the response (weight or nitrogen gain) evaluated as a function of nitrogen intake. The individual nitrogen intake-animal response results could be described by a series of curves using a four-parameter logistic model. The use of parameter sharing permitted the full range of responses to be described so that statistical differences between the dose-response curves could be identified. When used for either weight maintenance, nitrogen equilibrium, maximum weight gain or maximum nitrogen retention, the five protein sources could be ranked in the following order: LA greater than HCM greater than MCM = SP greater than TCM. Relative to lactalbumin, the value of all four protein sources decreased with increasing nitrogen intake. The low values obtained for TCM may have been related to factors other than protein quality. The results of this study indicate that some insect proteins are equivalent or superior to soy protein as a source of amino acids for growing rats.

Amino Acids↗

PubMed2003

Insect cell culture for industrial production of recombinant proteins.

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.

Animals↗

PubMedDate not supplied

Baculovirus-based expression of an insect viral protein in 12 different insect cell lines.

The ability of 12 unique lepidopteran insect cell lines from Anticarsia gemmatalis, Heliothis virescens, Lymantria dispar (two lines), Mamestra brassica, Plutella xylostella, Spodoptera frugiperda (two lines), and Trichoplusia ni (three lines) to support production of a recombinant polydnavirus (PDV) protein (GiPDV 1.1) expressed using the Bac-to-Bac baculovirus expression system was examined. Polydnavirus gene GiPDV 1.1 was cloned into the pFastBac baculovirus vector under the control of the polyhedron promoter, followed by generation of recombinant bacmid-GiPDV 1.1 by site-specific transposition. The ability of each insect cell line to support recombinant PDV gene expression was estimated using reverse transcriptase-polymerase chain reaction and Western blot. Each insect cell line infected with recombinant bacmid-GiPDV 1.1 and tested in this study was capable of supporting and producing recombinant protein. Time course expression analysis showed that 72-96 h after transfection to be the optimal time for harvest of recombinant protein for each insect cell line.

Animals↗

PubMed2000

Mimicry of ice structure by surface hydroxyls and water of a beta-helix antifreeze protein.

Insect antifreeze proteins (AFP) are much more effective than fish AFPs at depressing solution freezing points by ice-growth inhibition. AFP from the beetle Tenebrio molitor is a small protein (8.4 kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-A resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular beta-helix. Not only are the 12-amino-acid loops almost identical in the backbone, but also the conserved side chains are positioned in essentially identical orientations, making this AFP perhaps the most regular protein structure yet observed. The protein has almost no hydrophobic core but is stabilized by numerous disulphide and hydrogen bonds. On the conserved side of the protein, threonine-cysteine-threonine motifs are arrayed to form a flat beta-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation and the spacing between OH groups is a near-perfect match to the ice lattice. Together with tightly bound co-planar external water, three ranks of oxygen atoms form a two-dimensional array, mimicking an ice section.

Animals↗

PubMed2001

The Short antennae gene of Tribolium is required for limb development and encodes the orthologue of the Drosophila Distal-less protein.

Insects bear a stereotyped set of limbs, or ventral body appendages. In the highly derived dipteran Drosophila melanogaster, the homeodomain transcription factor encoded by the Distal-less (Dll) gene plays a major role in establishing distal limb structures. We have isolated the Dll orthologue (TcDll) from the beetle Tribolium castaneum, which, unlike Drosophila, develops well-formed limbs during embryogenesis. TcDll is initially expressed at the sites of limb primordia formation in the young embryo and subsequently in the distal region of developing legs, antennae and mouthparts except the mandibles. Mutations in the Short antennae (Sa) gene of Tribolium delete distal limb structures, closely resembling the Dll phenotype in Drosophila. TcDll expression is severely reduced or absent in strong Sa alleles. Genetic mapping and molecular analysis of Sa alleles also support the conclusion that TcDll corresponds to the Sa gene. Our data indicate functional conservation of the Dll gene in evolutionarily distant insect species. Implications for evolutionary changes in limb development are discussed.

Alleles↗

PubMed2002

Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems.

Insect cells are used routinely to express recombinant mammalian glycoproteins. However, insect protein glycosylation pathways are not well understood and appear to differ from those of mammalian cells. One way to more clearly evaluate the protein glycosylation potential of insect cells is to use the Drosophila melanogaster genome to identify genes that might encode relevant functions. These genes can then be expressed and the functions of the gene products directly evaluated by biochemical assays. In this study, we used this approach to determine the function of a putative Drosophila nucleotide sugar transporter gene. The results showed that this gene encodes a protein that can transport UDP-galactose, but not CMP-sialic acid. Thus, Drosophila encodes at least some of the infrastructure needed to produce glycoproteins with complex glycans, but this particular gene product does not directly support glycoprotein sialylation. These findings are relevant to insect cell biology and to an informed consideration of insect cell expression systems as tools for recombinant glycoprotein production.

Amino Acid Sequence↗

PubMed1994

Evolution of arthropod hemocyanins and insect storage proteins (hexamerins).

Crustacean and cheliceratan hemocyanins (oxygen-transport proteins) and insect hexamerins (storage proteins) are homologous gene products, although the latter do not bind oxygen and do not possess the copper-binding histidines present in the hemocyanins. An alignment of 19 amino acid sequences of hemocyanin subunits and insect hexamerins was made, based on the conservation of elements of secondary structure observed in X-ray structures of two hemocyanin subunits. The alignment was analyzed using parsimony and neighbor-joining methods. Results provide strong indications for grouping together the sequences of the 2 crustacean hemocyanin subunits, the 5 cheliceratan hemocyanin subunits, and the 12 insect hexamerins. Within the insect clade, four methionine-rich proteins, four arylphorins, and two juvenile hormone-suppressible proteins from Lepidoptera, as well as two dipteran proteins, form four separate groups. In the absence of an outgroup sequence, it is not possible to present information about the ancestral state from which these proteins are derived. Although this family of proteins clearly consists of homologous gene products, there remain striking differences in gene organization and site of biosynthesis of the proteins within the cell. Because studies on 18S and 12S rRNA sequences indicate a rather close relationship between insects and crustaceans, we propose that hemocyanin is the ancestral arthropod protein and that insect hexamerins lost their copper-binding capability after divergence of the insects from the crustaceans.

Amino Acid Sequence↗

PubMedDate not supplied

Facile and statistical optimization of transfection conditions for secretion of foreign proteins from insect Drosophila S2 cells using green fluorescent protein reporter.

Insect Drosophila melanogaster S2 cells were developed as a plasmid-based and therefore nonlytic expression system for functional foreign proteins. Transfection is an important step to introduce foreign target DNA into cells and should be properly optimized to obtain maximum production yield. Single factor search (SFS) methodology is still generally used to determine optimal condition in a biological system. Although this method is relatively simple to perform, it has many disadvantages such as not considering interactions between several factors and not covering the entire region of the solution pool. Therefore, we approached this optimization problem statistically with response surface (RSM) and evolutionary operation (EVOP) methodologies and compared the transfection efficiencies with the traditional SFS method. We employed secreted green fluorescent protein (GFP) as a reporter for determination of optimal transfection condition and secreted human erythropoietin (hEPO) as a confirming foreign model protein. Consequently, we arrived at the best optimal transient transfection condition (1 microg of plasmid DNA, 5 microg of lipofectin, 2 x 10(6) cells of initial cell number, and 18 h of transfection duration time) through a systematic access in a series of SFS, RSM, and EVOP. The secreted hEPO yield using optimal transient transfection condition by EVOP methodology was enhanced by about 1.8-fold compared to that of traditional SFS. This optimized transient transfection condition can be used as a basis for optimal stable transfections. A linear relationship between secreted GFP fluorescence intensity and secreted hEPO concentration indicated that facile and noninvasive determination of optimal transfection conditions for expression and secretion of foreign proteins in S2 cell cultures was made possible by simple measurement of GFP fluorescence.

Animals↗

PubMed1994

Human 5-HT1A receptor expressed in insect cells activates endogenous G(o)-like G protein(s).

Insect cell expression systems are used to characterize signaling components such as G protein-coupled receptors. As such, one must know whether endogenous G proteins couple to non-native receptors. We examined G protein linkages after infection of Sporodoptera frugiperda (Sf9) cells with a baculovirus encoding the 5-HT1A receptor. Receptor expression was confirmed by immunoblot. Some of the receptors were functional, showing guanine nucleotide-sensitive binding to the specific agonist ligand [3H]8-hydroxy-2-(di-n-propylamino)-1,2,3,4-tetranaphthalene). Peak expression (approximately 150 fmol/mg of membrane protein) was attained approximately 72-96 h post-infection. 5-HT-increased covalent binding of [32P]GTP-azidoanilide to a 40 kDa band, which was identified as a G protein by nucleotide blocking, Mg2+ dependence, and immunoblot and immunoprecipitation studies. The band comigrated with 1) pertussis toxin substrate(s), and 2) a band recognized by two G(o) alpha antisera and one common to heterotrimeric G protein alpha-subunits, but not by sera specific for Gs alpha or G(i) alpha. Labeled species could be precipitated with a G(o) alpha antiserum. 5-HT-increased labeling of the band was prevented by preincubation with pertussis toxin. These studies suggest that the 5-HT1A receptor couples effectively to native insect cell G(o)-like proteins.

Amino Acid Sequence↗

PubMed2003

A mini review of the high mobility group proteins of insects.

High mobility group (HMG) proteins are an abundant class of chromosomal proteins facilitate assembly of higher order structures. The mammalian HMG proteins have been grouped into three distinct families on the basis of their characteristic functional sequence: the HMGB, the HMGN, and the HMGA family. The HMG proteins of Drosophila melanogaster and Chironomus tentans are the best characterized dipteran insect HMG proteins. Three abundant members of this group of nonhistone proteins were detected in those insects. Two of them belong to the HMGB family and one to the HMGA family. The possible relatedness of these proteins to the formation of higher order nucleoprotein structures and their possible role in the regulation of transcription is discussed.

Amino Acid Motifs↗

PubMed1992

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.

Animals↗

PubMed1996

Overexpression of human nucleolar proteins in insect cells: characterization of nucleolar protein p120.

Nucleolar p120 is a proliferation-associated protein, which becomes detectable early in the G1 phase of the cell cycle and peaks early in the S phase. A variety of human malignant tumor cells contain much higher levels of p120 than normal resting cells. The cellular functions of p120 are unknown, and little information is available on the structural characteristics of the human p120 protein. For biochemical characterization, human p120 protein was expressed in a baculovirus system and purified to approximately 95% purity. By indirect immunofluorescence, most of the recombinant human p120 as well as recombinant human B23, C23, or fibrillarin were localized to insect cell nucleoli and to large globular nuclear inclusions. Like endogenous p120 in HeLa cells, recombinant p120 expressed in insect cells was phosphorylated. On sucrose density gradients, p120 from HeLa cells sedimented in the 60-80S region, in which preribosomal particles sedimented using similar extraction and centrifugation procedures. The sedimentation of p120 shifted to the 5-10S region by treatment with 1 M KCl or with RNAse which suggests that p120 is bound to RNA.

Amino Acid Sequence↗

PubMed1982

Isolation, characterization, and amino acid sequence of a ubiquitin-like protein from insect eggs.

A protein with a primary structure identical to that of human and bovine ubiquitin has been purified from insect eggs. The isolation, secondary structure, and amino acid sequence of this ubiquitin-like protein are reported. The sequence was determined by automatic Edman degradation of the intact molecule as well as by the manual sequence analysis of the enzymatic cleavage products. The polypeptide has 74 amino acid residues and internal homology regions. Interactions of the protein with peptides results in protective effects against proteolysis. This paper reports for the first time the presence of the ubiquitin molecule in invertebrates.

Amino Acid Sequence↗