| mechanism of activation of human heparanase investigated by protein engineering. | the aim of this study was to investigate the mechanism of activation of human heparanase, a key player in heparan sulfate degradation, thought to be involved in normal and pathologic cell migration processes. active heparanase arises as a product of a series of proteolytic processing events. upon removal of the signal peptide, the resulting, poorly active 65 kda species undergoes the excision of an intervening 6 kda fragment generating an 8 kda polypeptide and a 50 kda polypeptide, forming the f ... | 2004 | 14967027 |
| human influenza virus ns1 protein enhances viral pathogenicity and acts as an rna silencing suppressor in plants. | rna silencing has a well-established function as an antiviral defence mechanism in plants and insects. using an agrobacterium-mediated transient assay, we report here that ns1 protein from human influenza a virus suppresses rna silencing in plants in a manner similar to p1/hc-pro protein of tobacco etch potyvirus, a well-characterized plant virus silencing suppressor. moreover, we have shown that ns1 protein expression strongly enhances the symptoms of potato virus x in three different plant hos ... | 2004 | 15039541 |
| structural characterization of tobacco etch virus coat protein mutants. | the assembly of tobacco etch potyvirus (tev) coat protein (cp) and truncated mutants in escherichia coli was studied. cp from which 28, 63 or 112 amino acids were deleted from the n-terminus polymerized into potyvirus-like particles (pvlps). these structures were more rigid and progressively smaller in diameter than those produced by full length tev-cp. cp from which 175 n-terminal amino acids were removed, failed to polymerize. a fragment containing amino acids 131 to 206 of tev-cp is sufficien ... | 2004 | 15045558 |
| interaction of tobacco etch or tobacco vein mottling virus and ozone on biomass changes in burley tobacco. | burley tobacco is susceptible to several different types of virus diseases that suppress plant growth and development. two viruses, tobacco etch virus (tev) and tobacco vein mottling virus (tvmv), are particularly damaging to burley. burley tobacco cultivars resistant to these two viruses are currently being developed. some of these cultivars also show differential sensitivity to ozone (o3). recent field observations have suggested that burley tobacco infected with tev and tvmv was more sensitiv ... | 1988 | 15092551 |
| automated large-scale purification of a g protein-coupled receptor for neurotensin. | structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. here, we describe a protocol for the purification of a g protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. fermentation at a 200-l scale of escherichia coli expressing functional receptors provides the material needed to feed into the pur ... | 2004 | 15111111 |
| potyviral nia proteinase, a proteinase with novel deoxyribonuclease activity. | the nia proteinase from pepper vein banding virus (pvbv) is a sequence-specific proteinase required for processing of viral polyprotein in the cytoplasm. it accumulates in the nucleus of the infected plant cell and forms inclusion bodies. the function of this protein in the nucleus is not clear. the purified recombinant nia proteinase was active, and the mutation of the catalytic residues his-46, asp-81, and cys-151 resulted in complete loss of activity. most interesting, the pvbv nia proteinase ... | 2004 | 15163663 |
| functional replacement of wheat streak mosaic virus hc-pro with the corresponding cistron from a diverse array of viruses in the family potyviridae. | helper component-proteinase (hc-pro) of wheat streak mosaic virus strain sidney 81 (wsmv-sidney 81) was systematically replaced with the corresponding cistron derived from four strains of wsmv (type, tk1, cz, and el batán 3), the tritimovirus oat necrotic mottle virus (onmv), the rymoviruses agropyron mosaic virus (agmv) and hordeum mosaic virus (homv), or the potyviruses tobacco etch virus (tev) and turnip mosaic virus (tumv). these hc-pro proteins varied in amino acid sequence identity shared ... | 2004 | 15193921 |
| the type-1 and type-2 ribosome-inactivating proteins from iris confer transgenic tobacco plants local but not systemic protection against viruses. | the antiviral activity of the type-2 ribosome-inactivating protein (rip) irab from iris was analyzed by expressing irab in tobacco (nicotiana tabacum l. cv. samsun nn) plants and challenging the transgenic plants with tobacco mosaic virus (tmv). although constitutive expression of irab resulted in an aberrant phenotype, the plants were fertile. transgenic tobacco lines expressing irab showed a dose-dependent enhanced resistance against tmv infection but the level of protection was markedly lower ... | 2004 | 15278456 |
| structural analysis of the eukaryotic initiation factor 4e gene controlling potyvirus resistance in pepper: exploitation of a bac library. | the pvr2 locus in pepper codes for a eukaryotic translation initiation factor 4e (eif4e) gene that confers resistance to viruses belonging to the potyvirus genus. in this work, we describe the isolation and characterisation of the genomic sequence carrying the pvr2 locus. a bacterial artificial chromosome (bac) library that consisted of 239,232 clones with an average insert size of 123 kilobases (kb) was constructed from a capsicum annuum line with the pvr2(+) allele for susceptibility to potato ... | 2004 | 15315824 |
| heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of rna interference in drosophila cells. | rna interference (rnai) in animals and post-transcriptional gene silencing (ptgs) in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. plant viruses respond by expressing suppressor proteins that interfere with the ptgs system. | 2004 | 15331016 |
| over-expression of the human mdm2 p53 binding domain by fusion to a p53 transactivation peptide. | mdm2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells. although it is possible to prepare a small amount of the region of mdm2 that binds to p53, the expression level of this fragment of mdm2 is relatively low, limiting the studies involving this protein. here, we describe a construct for the optimized bacterial expression and purification of the mdm2 p53 binding domain. we found that the expression level of the soluble mdm2 p53 binding domain in bacteria was inc ... | 2004 | 15358376 |
| efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro. | affinity tags are widely used as vehicles for the production of recombinant proteins. yet, because of concerns about their potential to interfere with the activity or structure of proteins, it is almost always desirable to remove them from the target protein. the proteases that are most often used to cleave fusion proteins are factor xa, enterokinase, and thrombin, yet the literature is replete with reports of fusion proteins that were cleaved by these proteases at locations other than the desig ... | 2004 | 15477088 |
| [comparative analysis of virulence of the siberian and far-east subtypes of the tick-born encephalitis virus]. | the siberian subtype of the tick-borne encephalitis virus (tev) is different from the far-east subtype by a moderate virulence observed in siberian hamsters and by a low infection development rate (100 strains were compared). no differences were found in neuro-invasiveness. clinical findings and experiments with monkeys denote the ability of the siberian subtype to provoke severe forms of tick-borne encephalitis (tbe). the inflammation-and-degenerative changes were localized in the brain cortex, ... | 2004 | 15597957 |
| high level expression and single-step purification of hexahistidine-tagged l-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set. | affinity tags as fusions to the n- or c-terminal part of proteins are valuable tools to facilitate the production and purification of proteins. in many cases, there may be the necessity to remove the tag after protein preparation to regain activity. removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease. here, we report the construction of an expression vector set that combines n- or c-terminal fusion to either a ... | 2005 | 15642463 |
| a new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in e. coli as tev protease cleavable fusion protein. | we report here the use of tev protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins. bacterial expression was utilized to produce two fusion proteins, gprt-c37-h6 and his-tagged interleukin-2 (amino acids 6-133), which when cleaved by the tobacco etch virus nia protease (tev protease) to generate hiv entry inhibitor peptide c37-h6 and a truncated version of the cytokine interleukin-2, both containing n-terminal cysteines. the n-terminal cysteine containing c3 ... | 2005 | 15653356 |
| comparison of the substrate specificity of two potyvirus proteases. | the substrate specificity of the nuclear inclusion protein a (nia) proteolytic enzymes from two potyviruses, the tobacco etch virus (tev) and tobacco vein mottling virus (tvmv), was compared using oligopeptide substrates. mutations were introduced into tev protease in an effort to identify key determinants of substrate specificity. the specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. the crystal structure of tev protease and a homology model of t ... | 2005 | 15654889 |
| an alternative tandem affinity purification strategy applied to arabidopsis protein complex isolation. | tandem affinity purification (tap) strategies constitute an efficient approach for protein complex purification from many different organisms. however, the application of such strategies for purifying endogenous arabidopsis multi-protein complexes has not yet been reported. here, we describe an alternative tap (tapa) system that successfully allows protein complex purification from arabidopsis. in our newly generated tapa tag we have replaced the tobacco etch virus (tev) protease cleavage site w ... | 2005 | 15703063 |
| rapid, high-yield expression and purification of ca2+-atpase regulatory proteins for high-resolution structural studies. | phospholamban (plb) and sarcolipin (sln) are small integral membrane proteins that regulate the ca(2+)-atpases of cardiac and skeletal muscle, respectively, and directly alter their calcium transport properties. plb interacts with and regulates the cardiac ca(2+)-atpase at submaximal calcium concentrations, thereby slowing relaxation rates and reducing contractility in the heart. sln interacts with and regulates the skeletal muscle ca(2+)-atpase in a mechanism analogous to that used by plb. whil ... | 2005 | 15721779 |
| structural analysis of tobacco etch potyvirus hc-pro oligomers involved in aphid transmission. | oligomeric forms of the hc-pro protein of the tobacco etch potyvirus (tev) have been analyzed by analytical ultracentrifugation and single-particle electron microscopy combined with three-dimensional (3d) reconstruction. highly purified hc-pro protein was obtained from plants infected with tev by using a modified version of the virus that incorporates a histidine tag at the hc-pro n terminus (hishc-pro). the purified protein retained a high biological activity in solution when tested for aphid t ... | 2005 | 15731269 |
| self-cleavage of fusion protein in vivo using tev protease to yield native protein. | overproduction of proteins from cloned genes using fusion protein expression vectors in escherichia coli and eukaryotic cells has increased the quantity of protein produced. this approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. one major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additio ... | 2005 | 15741334 |
| target-directed proteolysis at the ribosome. | target directed proteolysis allows specific processing of proteins in vivo. this method uses tobacco etch virus (tev) nia protease that recognizes a seven-residue consensus sequence. because of its specificity, proteins engineered to contain a cleavage site are proteolysed, whereas other proteins remain unaffected. therefore, this approach can be used to study the structure and function of target proteins in their natural environment within living cells. one application is the conditional inacti ... | 2005 | 15784745 |
| high level expression of peptides and proteins using cytochrome b5 as a fusion host. | a novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. the ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase i (ilvm) and ii (ilvn), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins ... | 2005 | 15802225 |
| expression of a self-processing, pathogen resistance-enhancing gene construct in arabidopsis. | a gene cassette, p35s-cno, was designed to express three gene products driven by a single constitutive camv 35s promoter. the individual coding regions were linked in frame to produce a single polyprotein, using spacer sequences encoding a specific heptapeptide cleavage recognition site (enlyfqs) for the nuclear-inclusion-a (nia) proteinase of tobacco etch virus (tev). the protein coding sequences used were: a trichoderma harzinum endochitinase, a truncated nia proteinase of tev, and a wheat oxa ... | 2005 | 15834810 |
| the pvr1 locus in capsicum encodes a translation initiation factor eif4e that interacts with tobacco etch virus vpg. | mutations in the eif4e homolog encoded at the pvr1 locus in capsicum result in broad-spectrum potyvirus resistance attributed to the pvr1 resistance allele, a gene widely deployed in agriculture for more than 50 years. we show that two other resistance genes, previously known to be eif4e with narrower resistance spectra, pvr2(1) and pvr2(2), are alleles at the pvr1 locus. based on these data and current nomenclature guidelines, we have re-designated these alleles, pvr1(1) and pvr1(2), respective ... | 2005 | 15842624 |
| cap-independent translation of tobacco etch virus is conferred by an rna pseudoknot in the 5'-leader. | the tobacco etch virus (tev) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mrna, indicating that the leader contains an internal ribosome entry site. the tev 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an rna pseudoknot. mutational analysis of the tev 5'-leader identified pseudoknot (pk) 1 within the 5'-proximal domain and an ... | 2005 | 15911616 |
| crystal structure of tobacco etch virus protease shows the protein c terminus bound within the active site. | tobacco etch virus (tev) protease is a cysteine protease exhibiting stringent sequence specificity. the enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins. crystal structures of two tev protease mutants as complexes with a substrate and a product peptide provided the first insight into the mechanism of substrate specificity of this enzyme. we now report a 2.7a crystal structure of a full-length inactive c151a mutant protein crystallised i ... | 2005 | 15919091 |
| the recessive potyvirus resistance gene pot-1 is the tomato orthologue of the pepper pvr2-eif4e gene. | the translation initiation factor 4e (eif4e) has been implicated in naturally occurring resistance to potato virus y (pvy) determined by the pvr2 locus in pepper (capsicum annuum). here, the molecular basis of the recessive resistance to pvy and tobacco etch virus (tev) controlled by the pot-1 locus in tomato (lycopersicon esculentum; now solanum lycopersicum) was investigated. on the basis of genetic mapping data that indicated that pot-1 and pvr2 are located in syntenic regions of the tomato a ... | 2005 | 15971038 |
| a tandem orthogonal proteolysis strategy for high-content chemical proteomics. | the field of proteomics aims to assign functions to the numerous protein products encoded by eukaryotic and prokaryotic genomes. toward this end, chemical strategies have emerged as a powerful means to enrich specific classes of proteins based on shared functional properties, such as catalytic activity [activity-based protein profiling (abpp)], and post-translational modification state. the theoretical information content in chemical proteomic experiments greatly exceeds the actual data procured ... | 2005 | 16011363 |
| mediator and tfiih govern carboxyl-terminal domain-dependent transcription in yeast extracts. | in saccharomyces cerevisiae, the rna polymerase ii (rna pol ii) carboxyl-terminal domain (ctd) is required for viability, and truncation of the ctd results in promoter dependent transcriptional defects. a ctd-less rna pol ii is unable to support transcription in yeast extracts, but basal transcription reactions reconstituted from highly purified general transcription factors are ctd-independent. to reconcile these two findings, we have taken a biochemical approach using yeast extracts and asked ... | 2005 | 16076843 |
| plant pathology and rnai: a brief history. | this article describes the discovery of rna-activated sequence-specific rna degradation, a phenomenon now referred to as rna silencing or rna interference (rnai). from 1992 to 1996, a series of articles were published on virus resistant transgenic plants expressing either translatable or nontranslatable versions of the coat protein gene of tobacco etch virus (tev). certain transgenic plant lines were resistant to tev but not to closely related viruses. in these plants a surprising correlation wa ... | 2005 | 16078882 |
| characterization of resistance in transgenic nicotiana benthamiana encoding n-terminal deletion and assembly mutants of the tobacco etch potyvirus coat protein. | the resistance of transgenic nicotiana benthamiana plants encoding wild type, truncated and point mutants of the tobacco etch virus (tev) coat protein (cp) was analyzed. after r1 plants from 45 transgenic lines were challenged with tev, six percent of the lines exhibited high resistance, 38% exhibited low resistance, and the remainder were susceptible. the phenomenon of recovery and delay in symptom development was observed in 65% and 56% of the resistant and susceptible lines, respectively. pla ... | 2005 | 16086100 |
| improved solubility of tev protease by directed evolution. | the efficiency and high specificity of tobacco etch virus (tev) protease has made it widely used for cleavage of recombinant fusion proteins. however, the production of tev protease in e. coli is hampered by low solubility. we have subjected the gene encoding tev protease to directed evolution to improve the yield of soluble protein. libraries of mutated genes obtained by error-prone pcr and gene shuffling were introduced into the gateway cloning system for facilitated transfer between vectors f ... | 2006 | 16150509 |
| solubility-enhancing proteins mbp and nusa play a passive role in the folding of their fusion partners. | it is well established that certain highly soluble proteins have the ability to enhance the solubility of their fusion partners. however, very little is known about how different solubility enhancers compare in terms of their ability to promote the proper folding of their passenger proteins. we compared the ability of two well-known solubility enhancers, escherichia coli maltose-binding protein (mbp) and n utilization substance a (nusa), to improve the solubility and promote the proper folding o ... | 2006 | 16168669 |
| development of bean pod mottle virus-based vectors for stable protein expression and sequence-specific virus-induced gene silencing in soybean. | plant virus-based vectors provide valuable tools for expression of foreign proteins in plants and for gene function studies. none of the presently available virus vectors is suitable for use in soybean. in the present study, we produced bean pod mottle virus (bpmv)-based vectors that are appropriate for gene expression and virus-induced gene silencing (vigs) in soybean. the genes of interest were inserted into the rna2-encoded polyprotein open reading frame between the movement protein (mp) and ... | 2006 | 16226780 |
| an optimized system for expression and purification of secreted bacterial proteins. | in this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. candidate secreted proteins were produced recombinantly in escherichia coli as tobacco etch virus protease-cleavable hexahistidine-c-myc eptiope fusion proteins. without regard to their initial solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, purified under denaturing conditions by immobilized metal affinity ... | 2006 | 16260150 |
| highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination. | tandem affinity purification (tap) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. the method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. however, we found that the original method, which is based on the tap tag, consisting of a duplicate protein a epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (cbp), did not ... | 2005 | 16278461 |
| an improved expression plasmid for affinity purification of staphylococcus aureus gyrase a subunit. | of the bacterial topoisomerases, the gyrase a subunit (gyra) of staphylococcus aureus is particularly difficult to purify because of its tendency to form inclusion bodies. previous attempts at purification yielded low concentrations of protein with reduced specific activity. to overcome this problem, we modified the commercially available plasmid expression vector, pbad/thio-topo, via the addition of dna sequences encoding a hexahistidine tag upstream and a cleavage site for tobacco etch virus p ... | 2006 | 16289915 |
| expression and purification of sea raven type ii antifreeze protein from drosophila melanogaster s2 cells. | we present a system for the expression and purification of recombinant sea raven type ii antifreeze protein, a cysteine-rich, c-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. the cdnas encoding the pro- and mature forms of the sea raven protein were cloned into a modified pmt drosophila expression vector. these constructs produced n-terminally his(6)-tagged pro- and mature forms of the type ii antifreeze protein under the c ... | 2006 | 16330225 |
| improving solubility of shewanella oneidensis mr-1 and clostridium thermocellum jw-20 proteins expressed into esherichia coli. | low solubility of proteins overexpressed in e. coli is a frequent problem in high-throughput structural genomics. to improve solubility of proteins from mesophilic shewanella oneidensis mr-1 and thermophilic clostridium thermocellum jw20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (mbp) and a decrease of induction temperature. the mbp was selected as the most efficient solubilizing carrier when compared to a glutathione s-transferase and a ... | 2005 | 16335938 |
| spontaneous short-range silencing of a gfp transgene in nicotiana benthamiana is possibly mediated by small quantities of sirna that do not trigger systemic silencing. | a green fluorescent protein (gfp) transgene under the control of the 35s cauliflower mosaic virus (camv) promoter was introduced by agrobacterium-mediated transformation into nicotiana benthamiana to generate fourteen transgenic lines. homozygous lines that contained one or two copies of the transgene showed great variation of gfp expression under ultraviolet (uv) light, which allowed classification into three types of transgenic plants. plants from more than half of the transgenic lines underwe ... | 2006 | 16507090 |
| sequence determination of the capsid protein gene and flanking regions of tobacco etch virus: evidence for synthesis and processing of a polyprotein in potyvirus genome expression. | the nucleotide sequence of the 3'-terminal portion of the tobacco etch virus (tev) genome was determined. the 2324-nucleotide sequence represented approximately one-fourth of the tev genome and included the capsid protein gene and flanking regions. an open reading frame of 2135 nucleotides and an untranslated region of 189 nucleotides adjacent to a polyadenylate tract were identified. the sequence began within an open reading frame, indicating that the initiation codon was upstream of the availa ... | 1985 | 16593574 |
| pseudomonas aeruginosa porin oprf exists in two different conformations. | the major nonspecific porin of pseudomonas aeruginosa, oprf, produces a large channel yet allows only a slow diffusion of various solutes. here we provide an explanation of this apparent paradox. we first show, by introduction of tobacco etch virus protease cleavage site in the middle of oprf protein, that most of oprf population folds as a two-domain protein with an n-terminal beta-barrel domain and a c-terminal periplasmic domain rich in alpha-helices. however, sedimentation of unilamellar pro ... | 2006 | 16595653 |
| bacterial expression of functional, biotinylated peripheral cannabinoid receptor cb2. | a biotin-protein ligase recognition site (brs) was inserted into a polypeptide comprised of the maltose-binding protein, the peripheral cannabinoid receptor (cb2), thioredoxin a, and a polyhistidine tag at the carboxy terminus. expression levels of the recombinant receptor in escherichia coli bl21(de3) cells were approximately 1mg per liter of bacterial culture. the biotinylated cb2-fusion fully retained its ligand-binding capacity. introduction of the brs at the c-terminus of the cb2 fusion pro ... | 2006 | 16621595 |
| double-stranded rna binding may be a general plant rna viral strategy to suppress rna silencing. | in plants, rna silencing (rna interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. it is proposed that various suppressors inhibit rna silencing by targeting different steps. however, as double-stranded rnas (dsrnas) play key roles in silencing, it was speculated that dsrna binding might be a gene ... | 2006 | 16731914 |
| small nuclear inclusion protein encoded by a plant potyvirus genome is a protease. | tobacco etch virus, a plant potyvirus, expresses its rna genome as a large polyprotein precursor which undergoes extensive proteolytic processing to yield seven or more mature products. two of these products, proteins with apparent molecular weights of 49,000 and 54,000 (49k and 54k proteins), aggregate in the form of crystalline inclusions within the nuclei of infected cells. cell-free translation of synthetic transcripts was used to map the genes for these two products on the viral genome and ... | 1987 | 16789265 |
| incorporating a tev cleavage site reduces the solubility of nine recombinant mouse proteins. | failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. the expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. however, the additional residues can affect the properties of the protein; therefore, it is often desirabl ... | 2006 | 16798010 |
| a method for rapid protease substrate evaluation and optimization. | we have developed a high throughput assay for the measurement of protease activity in solution. this technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. the peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. protease cleavage causes the biotin containing fragment to be detached ... | 2006 | 16842230 |
| interaction of genome-linked protein (vpg) of turnip mosaic virus with wheat germ translation initiation factors eifiso4e and eifiso4f. | the interaction between vpg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eifiso4e and eifiso4f (the complex of eifiso4e and eifiso4g) were measured and compared. the fluorescence quenching data showed the presence of one binding site on eifiso4e for vpg. scatchard analysis revealed the binding affinity (k(a)) and average binding sites (n) for vpg were (8.51 +/- 0.21) x 10(6) m(-1) and 1.0, respectively. the addition of eifiso4g to the eifiso4e increased the bin ... | 2006 | 16880203 |
| an improved strategy for high-level production of tev protease in escherichia coli and its purification and characterization. | because of its stringent sequence specificity, tobacco etch virus (tev) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. however, the solubility of tev protease expressed in escherichia coli is extremely low. in the present study, we introduced a more efficient system to improve and facilitate the soluble production of tev protease in e. coli. optimal expression of soluble his6-tev was achieved by examining the contribution of chaperone c ... | 2007 | 16919473 |
| plasmid system for the intracellular production and purification of affinity-tagged proteins in bacillus megaterium. | a multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in bacillus megaterium was developed. the n- and c-terminal fusion of a protein of interest to a strep ii and a his(6)-tag is possible. corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. the exemplatory protein production of green fluorescent protein (gfp) showed differences in produced and recovered protein amou ... | 2007 | 16964623 |
| ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform. | myosin heavy chain (mhc) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. these kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament ca(2+) activation. to determine whether ca(2+) sensitivity of unloaded thin filament sliding depends upon mhc isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rshmm) or porcine cardiac myosin (pcmyosin). regulated t ... | 2006 | 17008370 |
| tobacco etch virus mrna preferentially binds wheat germ eukaryotic initiation factor (eif) 4g rather than eifiso4g. | the 5'-leader of tobacco etch virus (tev) genomic rna directs the efficient translation from the naturally uncapped viral rna. the tev 143-nt 5'-leader folds into a structure that contains two domains, each of which contains rna pseudoknots. the 5'-proximal pseudoknot 1 (pk1) is necessary to promote cap-independent translation (zeenko, v., and gallie, d. r. (2005) j. biol. chem. 280, 26813-26824). during the translation initiation of cellular mrnas, eif4g functions as an adapter that recruits ma ... | 2006 | 17012235 |
| salicylic acid-mediated and rna-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco. | plum pox virus (ppv) is able to replicate in inoculated leaves of nicotiana tabacum, but is defective in systemic movement in this host. however, ppv produces a systemic infection in transgenic tobacco expressing the silencing suppressor p1/hc-pro from tobacco etch virus (tev). in this work we show that ppv is able to move to upper non-inoculated leaves of tobacco plants expressing bacterial salicylate hydroxylase (nahg) that degrades salicylic acid (sa). replication and accumulation of ppv is h ... | 2006 | 17018032 |
| production of plum pox virus hc-pro functionally active for aphid transmission in a transient-expression system. | potyviruses are non-persistently transmitted by aphid vectors with the assistance of a viral accessory factor known as helper component (hc-pro), a multifunctional protein that is also involved in many other essential processes during the virus infection cycle. a transient agrobacterium-mediated expression system was used to produce plum pox virus (ppv) hc-pro in nicotiana benthamiana leaves from constructs that incorporated the 5' region of the genome, yielding high levels of hc-pro in agroinfi ... | 2006 | 17030878 |
| isolation of intracellular proteinase inhibitors derived from designed ankyrin repeat proteins by genetic screening. | the specific intracellular inhibition of protein activity at the protein level is a highly valuable tool for the validation or modulation of cellular processes. we demonstrate here the use of designed ankyrin repeat proteins (darpins) as tailor-made intracellular proteinase inhibitors. site-specific proteolytic processing plays a critical role in the regulation of many biological processes, ranging from basic cellular functions to the propagation of viruses. the nia(pro) proteinase of tobacco et ... | 2006 | 17050543 |
| monitoring regulated protein-protein interactions using split tev. | signaling cascades integrate extracellular stimuli primarily through regulated protein-protein interactions (ppis). intracellular signal transduction strictly depends on ppis occurring at the membrane and in the cytosol. to monitor constitutive and regulated protein interactions within living mammalian cells, we have developed a biological assay termed split tev. we engineered inactive fragments of the nia protease from the tobacco etch virus (tev protease) that regain activity only when coexpre ... | 2006 | 17072307 |
| a real-time rt-pcr assay for quantifying the fitness of tobacco etch virus in competition experiments. | relative fitness determination has become a standard tool in experimental virus evolution studies. in this type of studies, the tested strain is mixed with a reference strain, which differs in an easy-to-score and genetically stable marker, and allowed to compete for a limited common pool of resources during a given number of generations. in this report, a taqman real-time pcr methodology is proposed for quantifying the relative fitness of tobacco etch potyvirus strains (tev) in in planta mixed ... | 2007 | 17092574 |
| a co-localization assay for the analysis of protein-protein interactions. | the understanding and analysis of protein associations in living cells is a major goal of molecular biology. here, we describe an assay for the analysis of protein-protein interactions based on the co-localization of a fused site-specific protease with a cleavable reporter in close proximity to the interaction partner under examination. we exemplified this scheme in the temperature-sensitive saccharomyces cerevisiae cdc25-2 mutant strain using the nuclear inclusion protease of tobacco etch virus ... | 2007 | 17157449 |
| a fluorogenic substrate as quantitative in vivo reporter to determine protein expression and folding of tobacco etch virus protease in escherichia coli. | quantitative and folding reporters are adequate tools to optimize recombinant protein expression in various host organisms, including escherichia coli. to determine the yield of soluble active protease from the tobacco etch virus (tev), we developed a single-molecule assay based on the fluorogenic substrate ana-qs-mca. this substrate consists of a 10 amino acid peptide (enlyfqsgtk) containing the proteolytic cleavage sequence of the tev protease. the peptide works as a linker n-terminally tagged ... | 2007 | 17188891 |
| facile approach for constructing tev insertions to probe protein structure in vivo. | the tobacco etch virus (tev) protease has been used as a tool to examine protein structure in vivo. tev cleavage sites (tevcs) have been introduced via cloning into unique restriction sites or random transposon mutagenesis. we describe a facile, efficient method for introducing tevcs at precise locations in a gene to test specific predictions about protein structure. the method uses the lamda red recombination system to construct seamless, in-frame insertions of the tevcs at any desired location ... | 2006 | 17191617 |
| transient co-expression of post-transcriptional gene silencing suppressors and beta-glucuronidase in harvested lettuce leaf tissue does not improve recombinant protein accumulation in planta. | agrobacterium-mediated gene transfer was used to co-express three virus-derived post-transcriptional gene silencing (ptgs) suppressors, p19 from tomato bushy stunt virus and two species of helper component proteinase (hcpro) from tobacco etch virus (tev) and turnip mosaic virus, with beta-glucuronidase (gus) in harvested lettuce leaf tissue to investigate whether gus accumulation increases in the presence of ptgs suppressors. co-expression incubations were 3-5 days at 4 and 22 degrees c. gus act ... | 2007 | 17206371 |
| use of dual affinity tags for expression and purification of functional peripheral cannabinoid receptor. | the human peripheral cannabinoid receptor (cb2) was expressed as a fusion with the maltose-binding protein (at the n-terminus), thioredoxin a (at the c-terminus) and two small affinity tags (a strep-tag and a polyhistidine tag). expression levels of the recombinant receptor in escherichia coli bl21(de3) cells were dependent on location and type of tags in the expression construct, and were as high as 1-2mg per liter of bacterial culture. the recombinant receptor was ligand binding-competent, and ... | 2007 | 17223358 |
| a generic method for the production of recombinant proteins in escherichia coli using a dual hexahistidine-maltose-binding protein affinity tag. | a generic protocol that utilizes a dual hexahistidine-maltose-binding protein (his6-mbp) affinity tag has been developed for the production of recombinant proteins in escherichia coli. the mbp moiety improves the yield and enhances the solubility of the passenger protein while the his-tag facilitates its purification. the fusion protein (his6-mbp-passenger) is purified by immobilized metal affinity chromatography (imac) on nickel-nitrilotriacetic acid (ni-nta) resin and then cleaved in vitro wit ... | 2007 | 17272834 |
| purification of components of the translation elongation factor complex of plasmodium falciparum by tandem affinity purification. | plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. a major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. one approach to assign function to these proteins is ... | 2007 | 17307963 |
| fitness declines in tobacco etch virus upon serial bottleneck transfers. | it has been well established that populations of rna viruses transmitted throughout serial bottlenecks suffer from significant fitness declines as a consequence of the accumulation of deleterious mutations by the onset of muller's ratchet. bottlenecks are unavoidably linked to different steps of the infectious cycle of most plant rna viruses, such as vector-mediated transmissions and systemic colonization of new leaves. here we report evidence for fitness declines by the accumulation of deleteri ... | 2007 | 17344305 |
| target-directed proteolysis in vivo. | the experimental problems associated with in vivo studies of essential proteins or integral membrane proteins have triggered geneticists to generate novel approaches that have often led to insights of general relevance (shuman and silhavy, 2003). in order to extend the experimental portfolio, we developed target-directed proteolysis (tdp), an in vivo method allowing structural and functional characterization of target proteins in living cells. tdp is based on the activity of the highly sequence- ... | 2007 | 17352916 |
| new vectors for co-expression of proteins: structure of bacillus subtilis scoab obtained by high-throughput protocols. | the bacillus subtilis genes scoa and scob encode subunits of the heteromeric enzyme scoab, a putative succinyl-coa:acetoacetate coenzyme a transferase. high-throughput, ligation-independent cloning (lic) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. transfer of the lic region of vector pmcsg7 (l. stols, m. gu, l. dieckman, r. raffen, f.r. collar ... | 2007 | 17363272 |
| a generic protocol for the expression and purification of recombinant proteins in escherichia coli using a combinatorial his6-maltose binding protein fusion tag. | we describe a generic protocol for the overproduction and purification of recombinant proteins in escherichia coli. the strategy utilizes a dual his6-maltose binding protein (hismbp) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. the mbp moiety serves to enhance the solubility and promote the proper folding of its fusion partners, and the polyhistidine tag facilitates its purification to homogeneity. ... | 2007 | 17406599 |
| ectopic expression of a recessive resistance gene generates dominant potyvirus resistance in plants. | despite long-standing plant breeding investments and early successes in genetic engineering, plant viral pathogens still cause major losses in agriculture worldwide. early transgenic approaches involved the expression of pathogen-derived sequences that provided limited protection against relatively narrow ranges of viral pathotypes. in contrast, this study demonstrates that the ectopic expression of pvr1, a recessive gene from capsicum chinense, results in dominant broad-spectrum potyvirus resis ... | 2007 | 17511813 |
| a combined approach to improving large-scale production of tobacco etch virus protease. | tobacco etch virus nia proteinase (tev protease) is an important tool for the removal of fusion tags from recombinant proteins. production of tev protease in escherichia coli has been hampered by insolubility and addressed by many different strategies. however, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. here, we use a quantitative, high-throughput assay for tev protease activity in cell lysates t ... | 2007 | 17543538 |
| expression of a recombinant human sperm-agglutinating mini-antibody in tobacco (nicotiana tabacum l.). | the murine monoclonal antibody (mab) s19 recognizes an n-linked carbohydrate antigen designated sperm agglutination antigen-1 (saga1) located on the membrane protein cd52. this antigen is added to the sperm surface during epididymal maturation. binding of the s19 mab to saga-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. previous studies indicated that the s19 mab may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current sp ... | 2007 | 17566292 |
| genetic analysis of the short splice variant of the inhibitor of caspase-activated dnase (icad-s) in chicken dt40 cells. | we have studied the regulation of the caspase-activated dnase (cad) by its inhibitor, icad. to study the role of icad short and long splice forms icad-s and icad-l, respectively, in vivo, we constructed chicken dt40 cell lines in which the entire coding regions of icad alone or icad plus cad were deleted. icad and icad/cad double knock-outs lacked both dna fragmentation and nuclear fragmentation after the induction of apoptosis. we constructed a model humanized system in which human icad-l and c ... | 2007 | 17616520 |
| engineered apoptotic nucleases for chromatin research. | we have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal dna cleavage, dna fragmentation factor (dff). normally, in its inactive form, dff is a heterodimer composed of a 45-kda chaperone inhibitor subunit (dff45 or icad), and a 40-kda latent endonuclease subunit (dff40 or cad). upon caspase-3 cleavage of dff45, dff40 forms active endonuclease homo-oligomers. although saccharomyces ... | 2007 | 17626049 |
| large-scale overexpression and purification of adars from saccharomyces cerevisiae for biophysical and biochemical studies. | many biochemical and biophysical analyses of enzymes require quantities of protein that are difficult to obtain from expression in an endogenous system. to further complicate matters, native adenosine deaminases that act on rna (adars) are expressed at very low levels, and overexpression of active protein has been unsuccessful in common bacterial systems. here we describe the plasmid construction, expression, and purification procedures for adars overexpressed in the yeast saccharomyces cerevisi ... | 2007 | 17662848 |
| translation-coupled translocation of yeast fumarase into mitochondria in vivo. | fumarase represents proteins that cannot be imported into mitochondria after the termination of translation (post-translationally). utilizing mitochondrial and cytosolic versions of the tobacco etch virus (tev) protease, we show that mitochondrially targeted fumarase harboring a tev protease recognition sequence is efficiently cleaved by the mitochondrial but not by the cytosolic tev protease. nonetheless, fumarase was readily cleaved by cytosolic tev when its import into mitochondria was slowed ... | 2007 | 17666392 |
| predominant interaction of both ikaros and helios with the nurd complex in immature thymocytes. | ikaros is the founding member of a small family of c2h2 zinc-finger dna-binding proteins that carry out critical functions during lymphocyte development. although interactions between ikaros and various proteins have been reported, ikaros-containing complexes have not been purified to determine their composition and identify the predominant interacting partners. in this study, a tandem affinity purification-mass spectrometry strategy was developed for the isolation of complexes formed by ikaros ... | 2007 | 17681952 |
| purification and characterization of rgd tumor-homing peptide conjugated human tumor necrosis factor alpha over-expressed in escherichia coli. | a number of approaches have been investigated to enhance the selective toxicity of tumor necrosis factor alpha (tnfalpha) to permit its systemic use in cancer therapy. because vascular targeting has been proven to be a valid strategy for improving the therapeutic index of tnfalpha, we prepared rgd-htnf consisting of human tnf fused with the acdcrgdcfcg peptide, a ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins. recombinant rgd-htnf was produced in escherichia coli as a polyhistidine fusi ... | 2007 | 17716959 |
| a protein structure initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles. | a specialized vector backbone from the protein structure initiative was used to express full-length human cytochrome b5 as a c-terminal fusion to his8-maltose binding protein in escherichia coli. the fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3mg per g of wet weight bacterial cells). in situ proteolysis of the fusion protein in the presence of ... | 2008 | 18226920 |
| construction and use of new cloning vectors for the rapid isolation of recombinant proteins from escherichia coli. | we describe the construction and use of two sets of vectors for the over-expression and purification of protein from escherichia coli. the set of ptev plasmids (ptev3, 4, 5) directs the synthesis of a recombinant protein with a n-terminal hexahistidine (his(6)) tag that is removable by the tobacco etch virus (tev) protease. the set of pkld plasmids (pkld66, 116) directs the synthesis of a recombinant protein that contains a n-terminal his(6) and maltose-binding protein tag in tandem, which can a ... | 2008 | 18295882 |
| bifunctional nanotube scaffolds for diverse ligands are purified simply from escherichia coli strains coexpressing two functionalized flagellar genes. | we functionalized escherichia coli flic flagellin proteins to form tailored nanotubes binding single types or pairs of ligands, including divalent cations, fluorescent antibodies, or biotin-avidin-linked moieties such as ferritins. the ratio of each tag in bifunctionalized flagella could be toggled extending their sophistication as nanoscaffolds. tobacco etch virus (tev) protease site-containing flics were cleaved by the cognate protease without filament disintegration, potentiating their use as ... | 2007 | 17489638 |
| cylindrical inclusions in the cytoplasm of leaf cells infected with tobacco etch virus. | combined tracings from electron micrographs of serial sections of leaf tissue infected with tobacco etch virus show that one type of cytoplasmic inclusion, when sectioned in different planes, can produce configurations which have been interpreted as being two distinct types of inclusion bodies. | 1966 | 17780650 |
| functional dissection of naturally occurring amino acid substitutions in eif4e that confers recessive potyvirus resistance in plants. | naturally existing variation in the eukaryotic translation initiation factor 4e (eif4e) homolog encoded at the pvr1 locus in capsicum results in recessively inherited resistance against several potyviruses. previously reported data indicate that the physical interaction between capsicum-eif4e and the viral genome-linked protein (vpg) is required for the viral infection in the capsicum-tobacco etch virus (tev) pathosystem. in this study, the potential structural role(s) of natural variation in th ... | 2007 | 17890375 |
| distribution of fitness and virulence effects caused by single-nucleotide substitutions in tobacco etch virus. | little is known about the fitness and virulence consequences of single-nucleotide substitutions in rna viral genomes, and most information comes from the analysis of nonrandom sets of mutations with strong phenotypic effect or which have been assessed in vitro, with their relevance in vivo being unclear. here we used site-directed mutagenesis to create a collection of 66 clones of tobacco etch potyvirus, each carrying a different, randomly chosen, single-nucleotide substitution. competition expe ... | 2007 | 17898073 |
| enhancing the stability and solubility of tev protease using in silico design. | the ability to rationally increase the stability and solubility of recombinant proteins has long been a goal of biotechnology and has significant implications for biomedical research. poorly soluble enzymes, for example, result in the need for larger reaction volumes, longer incubation times, and more restricted reaction conditions, all of which increase the cost and have a negative impact on the feasibility of the process. rational design is achieved here by means of the popmusic program, which ... | 2007 | 17905838 |
| a multidirectional non-cell autonomous control and a genetic interaction restricting tobacco etch virus susceptibility in arabidopsis. | viruses constitute a major class of pathogens that infect a variety of hosts. understanding the intricacies of signaling during host-virus interactions should aid in designing disease prevention strategies and in understanding mechanistic aspects of host and pathogen signaling machinery. | 2007 | 17912362 |
| specific and efficient cleavage of fusion proteins by recombinant plum pox virus nia protease. | site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. nuclear inclusion protein a (nia) proteases obtained from the family potyviridae have become promising due to their high activities and stringencies of sequences recognition. nia proteases from tobacco etch virus (tev) and tomato vein mottling virus (tvmv) have been shown to process recombinant proteins successfully in vitro. in this report, recombinant ppv (plum pox virus) nia p ... | 2008 | 18024078 |
| potyvirus genome-linked protein, vpg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eif4f and eifiso4f. | potyvirus genome linked protein, vpg, interacts with translation initiation factors eif4e and eifiso4e, but its role in protein synthesis has not been elucidated. we show that addition of vpg to wheat germ extract leads to enhancement of uncapped viral mrna translation and inhibition of capped viral mrna translation. this provides a significant competitive advantage to the uncapped viral mrna. to understand the molecular basis of these effects, we have characterized the interaction of vpg with e ... | 2008 | 18045881 |
| incorporation of a polypeptide segment into the beta-domain pore during the assembly of a bacterial autotransporter. | bacterial autotransporters consist of an n-terminal 'passenger domain' that is transported into the extracellular space by an unknown mechanism and a c-terminal 'beta-domain' that forms a beta-barrel in the outer membrane. recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the beta-domain. it is unclear, however, whether this configuration forms prior to passenger domain translocat ... | 2008 | 18047580 |
| tom20 and tom22 share the common signal recognition pathway in mitochondrial protein import. | precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of tom20, tom22, and tom70 on the mitochondrial surface. tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. the cytosolic domain of tom22 appears to function as a receptor in cooperation with tom20, but how its substrate specificity differs from that of tom20 remains ... | 2008 | 18063580 |
| natural variation and functional analyses provide evidence for co-evolution between plant eif4e and potyviral vpg. | amino acid substitutions in the eukaryotic translation initiation factor 4e (eif4e) result in recessive resistance to potyviruses in a range of plant species, including capsicum spp. correspondingly, amino acid changes in the central part of the viral genome-linked protein (vpg) are responsible for the potyvirus's ability to overcome eif4e-mediated resistance. a key observation was that physical interaction between eif4e and the vpg is required for viral infection, and eif4e mutations that cause ... | 2008 | 18182024 |
| development of a method for expression and purification of the regulatory c2-like domain of human 5-lipoxygenase. | 5-lipoxygenase (5-lo), the key enzyme in leukotriene biosynthesis, is built of a catalytic c-terminal domain and a regulatory n-terminal c2-like domain. the c2-like domain is the target of many regulatory factors or proteins including ca(2+), phospholipids, glycerides, coactosin-like protein and presumably other components that modulate the catalytic activity of 5-lo by acting at this domain, but the detailed underlying molecular mechanisms of these interactions are still unclear. in order to ob ... | 2008 | 18280752 |
| identification of plant virus ires. | plant rna viruses exploit nonorthodox strategies, such as the use of internal ribosomal entry sites (ires), to express multiple genes from a single rna species. ires elements have been reported in tobacco etch virus (tev), crucifer infecting tobamovirus (crtmv), hibiscus chlorotic ringspot virus (hcrsv), and many other animal and plant rna viruses. in this chapter, the methodology used to identify and characterize a plant virus ires element, including construction of a translation reporter vecto ... | 2008 | 18370252 |
| biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (cb2(271-326)). | obtaining sufficient amount of purified g-protein coupled receptors (gpcrs) is almost always one of the major challenges for their structural studies. cb2(271-326), a human cannabinoid receptor 2 (cb2) fragment comprising part of the third extracellular loop (el3), the seventh transmembrane domain (tm7) and c-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (ib) of escherichia coli. the fusion protein was purified by histidine-selected nic ... | 2008 | 18375143 |
| identification of intracellular proteins associated with the ebv-encoded nuclear antigen 5 using an efficient tap procedure and ft-icr mass spectrometry. | epstein-barr virus nuclear antigen 5 (ebna5) is one of the first viral proteins detected after primary ebv infection and has been shown to be required for efficient transformation of b lymphocytes. ebna5 is a protein that has many suggested functions but the underlying biology remains to be clarified. to gain further insight into the biological roles of the proposed multifunctional ebna5, we isolated ebna5 containing protein complexes using a modified tandem affinity purification (tap) method an ... | 2008 | 18457437 |
| tev protease-mediated cleavage in drosophila as a tool to analyze protein functions in living organisms. | drosophila provides a powerful experimental system to analyze gene functions in a multi-cellular organism. here we describe an in vivo method that interferes with the integrity of selected proteins through site-specific cleavage in drosophila. the technique is based on the highly specific seven-amino-acid recognition site of the tobacco etch virus (tev) protease. we established transgenic fly lines that direct tev protease expression in various tissues without affecting fly viability. the insert ... | 2008 | 18476830 |
| inhibition of 3' modification of small rnas in virus-infected plants require spatial and temporal co-expression of small rnas and viral silencing-suppressor proteins. | plant viruses are inducers and targets of rna silencing. viruses counteract with rna silencing by expressing silencing-suppressor proteins. many of the identified proteins bind sirnas, which prevents assembly of silencing effector complexes, and also interfere with their 3' methylation, which protects them against degradation. here, we investigated the 3' modification of silencing-related small rnas in nicotiana benthamiana plants infected with viruses expressing rna silencing suppressors, the p ... | 2008 | 18539609 |
| quantification and extension of transient gfp expression by the co-introduction of a suppressor of silencing. | using particle bombardment, a dna expression vector containing the green fluorescent protein (gfp) reporter gene was introduced into plant cells. expression of the gfp gene was transient; resulting in peak gfp expression about 24 h post introduction and a rapid decline thereafter. this well known decline in gene expression has previously been attributed to pre-integrative dna events that involved the loss of introduced dna or cell death. here, we show that post-transcriptional gene silencing (pt ... | 2008 | 18548328 |
| in situ cleavage of the acidic domain from the p115 tether inhibits exocytic transport. | golgins are coiled-coil proteins involved in golgi architecture and function. a complex of golgins (p115, gm130 and giantin), together with the rab1 guanosine triphosphatase and cis golgi snares, helps to mediate fusion processes at the entry face of the golgi apparatus. the c-terminal acidic domain of p115 binds specifically to gm130 and giantin. however, deletion of this domain in vivo appears to have no effect on exocytic transport when using an rna interference depletion/rescue approach (put ... | 2008 | 18564369 |
| expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in japanese flounder, paralichthys olivaceus. | the cysteine-rich peptide hepcidin is an antimicrobial peptide and iron transport regulator that has been found in vertebrates including birds, fish and mammals. to elucidate the structure and biological function of fish hepcidin, which is difficult to produce synthetically, we have cloned several plasmid constructs encoding hepcidin from japanese flounder, paralichthys olivaceus, and tested expression of recombinant peptides, each with an n-terminal hexahistidine (6xhis) tag, in inclusion bodie ... | 2008 | 18595734 |
| hyper-acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in escherichia coli. | high expression of recombinant proteins in escherichia coli (e. coli) often leads to protein aggregation. one popular approach to address this problem is the use of fusion tags (or partners) that improve the solubility of the proteins in question. however, such fusion tags are not effective for all proteins. in this study, we demonstrate that the hyper-acidic protein fusion partners can largely enhance the soluble expression of target proteins recalcitrant to the efforts by using routine solubil ... | 2008 | 18599143 |