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At least 217 records · Page 12Linked to original sources
PubMed2004

Isolation and characterization of a novel rice gene encoding a putative insect-inducible protein homologous to wheat Wir1.

A full-length cDNA, designated BpHi008A, was cloned representing a rice (Oryza sativa) mRNA that accumulates after brown planthopper (BPH) Nilapar vata lugens Stål feeding. The cDNA encodes a putative 82 amino acid protein (BpHi008A) exhibiting about 37% amino acid sequence identity to Wir1 family of proteins that are encoded by pathogen-induced transcripts in wheat. Like Wir1 proteins, it consists of a hydrophobic N-terminal half and a hydrophilic C-terminal half relatively rich in glycine and proline. These proteins are predicted to be integrated into the membrane, with the C-terminus being extracytoplastic. Genomic Southern analysis indicated that the BpHi008A gene was present as a single-copy sequence in the rice genome. Temporal and spatial studies showed that BpHi008A were systemically induced in rice when 2nd and 3rd-instars were feeding. The BpHi008A transcripts level was also increased in seedlings damaged by mechanical wounding. These data indicated that BphHi008A was implicated in the response of rice plants to BPH feeding and wounding.

Amino Acid Sequence↗
PubMed2000

Molecular cloning and characterization of SRAM, a novel insect rel/ankyrin-family protein present in nuclei.

Previously, we purified a 59-kDa protein that binds to the kappaB motif of the Sarcophaga lectin gene. Here we report its cDNA cloning and some of its characteristics as a novel member of the Rel/Ankyrin-family. This protein, named SRAM, contained a Rel homology domain, a nuclear localization signal and 4 ankyrin repeats, but lacked the Ser-rich domain and PEST sequence that Relish contained. We found that SRAM was localized in the nuclei of NIH-Sape-4 cells, which are an embryonic cell line of Sarcophaga. The Sarcophaga lectin gene promoter containing tandem repeats of the kappaB motifs was activated in NIH-Sape-4 cells. In Drosophila mbn-2 cells, Dif alone activated this reporter gene and a cooperative effect was detected when SRAM and Dif were co-transfected, although SRAM alone did not activate it. This is the first report of a Rel/Ankyrin molecule that exists in the nuclei.

Amino Acid Sequence↗
PubMed1990

Characterization and purification of human fos protein generated in insect cells with a baculoviral expression vector.

We generated recombinant baculoviruses that contained the human fos gene and that, upon infection of insect cells, synthesized fos protein. The quantity of fos protein produced was at least 10 to 20 times higher than that observed in any mammalian cells reported so far. The fos protein made in insect cells manifested most of the characteristics of mammalian fos protein, which include (i) 55-kilodalton size, (ii) nuclear localization, (iii) phosphoesterification at serine residues, (iv) identical 35S tryptic peptide maps, (v) ability to make heterodimers with the nuclear jun oncoprotein, and (vi) cooperation with the jun protein to bind to a 12-O-tetradecanoyl-phorbol-13-acetate-responsive element. A 100- to 150-fold purification of the fos protein from infected insect cells was achieved in a single step by immunoaffinity chromatography. Availability of authentic fos protein made by baculoviral vectors in insect cells should allow a more rigorous analysis of its biochemical and biological properties.

Amino Acids↗
PubMed1997

Stable insect cell cultures for recombinant protein production.

Insect cells are relatively cheap to maintain and are capable of producing accurately translated and correctly processed heterologous proteins. Recent research has focused on the development of improved expression vectors for continuous, high-level production of foreign proteins, including a number of membrane-targeted receptors, in Drosophila and lepidopteran insect cells. Mosquito cells have also been employed for studies on the control of vector-borne diseases, such as malaria.

Animals↗
PubMed1995

Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein.

We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironomus tentans. Bacterially synthesized rCAS was purified to near homogeneity and characterized by several biochemical and biophysical methods including amino-terminal sequencing, amino acid compositional analysis, sedimentation equilibrium ultracentrifugation, and mass spectrometry. Complementing these techniques with quantitative sulfhydryl assays, we discovered that the four cysteines present in rCAS form two intramolecular disulfide bonds. Mapping studies revealed that the disulfide bonds are heterogeneous. When reduced and denatured rCAS was allowed to refold and its disulfide bonding state monitored, it again adopted a conformation with two intramolecular disulfide bonds. The inherent ability of rCAS to quantitatively form two intramolecular disulfide bonds may reflect a previously unknown feature of the in vivo silk proteins from which it is derived.

Amino Acid Sequence↗
PubMed2001

Two kinds of BIR-containing protein - inhibitors of apoptosis, or required for mitosis.

The baculoviral IAP repeat (BIR) is a zinc-binding fold. Some BIR-containing proteins (BIRCs), including several from insect viruses, insects and vertebrates, are inhibitors of cell death and act by binding to active caspases. Their ability to do so can be antagonized by pro-apoptotic insect proteins such as Grim, HID and Reaper, or the mammalian protein Diablo/Smac. Members of one structurally distinct subgroup of BIR-containing proteins, which are present in yeasts and Caenorhabditis elegans as well as insects and vertebrates, do not act as caspase inhibitors; instead, they are required for chromosome segregation and cytokinesis, and act in concert with inner centromere protein (INCENP) homologues and aurora kinase homologues.

Animals↗
PubMed1990

Insect immunity. Characterization of a Drosophila cDNA encoding a novel member of the diptericin family of immune peptides.

Drosophila shows an immune response when challenged by injection of low doses of bacteria. To date, the molecules involved in this immune reaction have remained elusive, with the exception of cecropins (4-kDa antibacterial peptides initially isolated from the moth Hyalophora cecropia) for which three closely related genes have been characterized recently. We report the molecular cloning and sequencing of a cDNA from a library of immune Drosophila which encodes a novel member of the family of diptericins (9-kDa antibacterial peptides initially isolated from the fly Phormia terranovae). Transcripts for the Drosophila diptericin are detected 2 h after injection of bacteria. They are apparently derived from a single gene mapping at position 56 A on the right arm of the second chromosome. We discuss the existence of a distant relationship between the diptericins and two other groups of anti-bacterial insect proteins, the attacins, and the sarcotoxins II.

Amino Acid Sequence↗
PubMed1994

Characterization of the Ras-related RAP2A protein expressed in the baculovirus-insect cell system: processing of the protein in insect cells and comparison with the bacterially produced unprocessed form.

The Ras-related protein Rap2A is a 21kD GTP-binding protein that exhibits 46% identity with Ras proteins and is similarly post-translationally modified by farnesyl and palmitate groups. Using a recombinant baculovirus, we expressed Rap2A in Sf9 insect cells. The protein is initially synthesized as a soluble hydrophilic precursor, that is post-translationally processed to a hydrophobic membrane-bound form (Rap2Am) that contains both isoprenoid and palmitate groups. The processed form of the protein was purified from the membranes of infected Sf9 cells, and its biochemical properties were compared with those of the unprocessed form produced in recombinant bacteria (Rap2Ab). Both proteins exhibited similar kinetics of GDP dissociation and GTP binding and displayed a weak intrinsic GTPase activity that was stimulated to the same extent by a factor present in bovine brain cytosol. We conclude that Rap2A is correctly processed in insect cells and that maturation does not alter its biochemical properties.

Animals↗
PubMed2003

High mobility group-like proteins of the insect Plodia interpunctella.

Nuclei from Plodia interpunctella larvae contain four major proteins, which are extracted by 5% perchloric acid and 0.35 M NaCl. The proteins have been designated PL1, PL2, PL3, and PL4. The amino acid analyses of these proteins show that they have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group (HMG) proteins isolated from vertebrate tissues. Immunological characterication of these proteins clearly shows that PL1, PL2, and PL4 are more closely related to HMG1 dipteran proteins, while PL3 is more closely related to HMG1 dipteran proteins. The possible relatedness of these proteins to HMG proteins is discussed.

Amino Acids↗
PubMed2005

Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells.

Rapid expression of recombinant proteins for structure determination is one of the major challenges in pharmaceutical and academic research, since the number of potential drug targets has increased significantly in the last decade. Despite the fact that the baculovirus expression vector system is widely used for this purpose, the system is hampered by three very slow and tedious procedures, namely generation of high titer baculovirus stock, determination of the virus titer and discovery of the best conditions for protein expression. We herein describe the development of the ultraBac system to address and overcome these issues for protein expression in insect cells. We have established a new baculovirus expression technology for insect cells that is based on co-expression of GFP with target genes, a new regime for cell culturing and a highly efficient purification and enrichment procedure for recombinant baculovirus particles. Co-expression of GFP is used to monitor the infection of insect cells, to simplify titer determination and to optimize expression conditions. The new regime for cell culturing with increased viability of non-infected insect cells and its combination with the massive enrichment of virus particles via high-speed centrifugation enables the production of large amounts of recombinant virus in a very short period of time. By combining these techniques and by using the bicistronic vector pUltraBac-1, we have been able to cut the time-lines for protein expression in insect cells by half, approaching those for protein production in Escherichia coli. This new expression system is a significant step forward towards industrialized protein production in both, industry and academia.

Animals↗
PubMed1998

Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein.

The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10-30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All eight cysteine residues appear to be involved in disulfide bond formation. Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive beta structure (70-80%) with little or no alpha helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0 degrees C to 20 degrees C), and the presence of 50% trifluoroethanol.

Amino Acid Sequence↗
PubMed1997

Role of lipid polymorphism in G protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes.

Heterotrimeric G proteins (peripheral proteins) conduct signals from membrane receptors (integral proteins) to regulatory proteins localized to various cellular compartments. They are in excess over any G protein-coupled receptor type on the cell membrane, which is necessary for signal amplification. These facts account for the large number of G protein molecules bound to membrane lipids. Thus, the protein-lipid interactions are crucial for their cellular localization, and consequently for signal transduction. In this work, the binding of G protein subunits to model membranes (liposomes), formed with defined membrane lipids, has been studied. It is shown that although G protein alpha-subunits were able to bind to lipid bilayers, the presence of nonlamellar-prone phospholipids (phosphatidylethanolamines) enhanced their binding to model membranes. This mechanism also appears to be used by other (structurally and functionally unrelated) peripheral proteins, such as protein kinase C and the insect protein apolipophorin III, indicating that it could constitute a general mode of protein-lipid interactions, relevant in the activity and translocation of some peripheral (amphitropic) proteins from soluble to particulate compartments. Other factors, such as the presence of cholesterol or the vesicle surface charge, also modulated the binding of the G protein subunits to lipid bilayers. Conversely, the binding of G protein-coupled receptor kinase 2 and the G protein beta-subunit to liposomes was not increased by hexagonally prone lipids. Their distinct interactions with membrane lipids may, in part, explain the different cellular localizations of all of these proteins during the signaling process.

Animals↗