| translation of potyvirus rna in a rabbit reticulocyte lysate: reaction conditions and identification of capsid protein as one of the products of in vitro translation of tobacco etch and pepper mottle viral rnas. | rnas of pepper mottle virus (pemv) and tobacco etch virus (tev) were efficient messengers when translated in the rabbit reticulocyte lysate system. viral rna (39 s) isolated from sucrose density gradients stimulated [35s]methionine incorporation into products precipitated by trichloroacetic acid 15- to 20-fold over endogenous background levels. optimal reaction conditions for in vitro translation contained 2 mug rna/30 mul reaction assay, 0.8-1.0 mm magnesium ions, and 100-125 mm potassium ions. ... | 1980 | 18631642 |
| translation of potyvirus rna in a rabbit reticulocyte lysate: identification of nuclear inclusion proteins as products of tobacco etch virus rna translation and cylindrical inclusion protein as a product of the potyvirus genome. | the translations of tobacco etch virus (tev) rna and pepper mottle virus (pemv) rna in the mrna-dependent rabbit reticulocyte lysate produced products which comigrated with potyviral inclusion proteins on polyacrylamide gels. the tev rna translation products comigrating with the 49,000 and 54,000 molecular weight tev nuclear inclusion proteins were also immunoprecipitated by antisera specific to the nuclear inclusion proteins. the proteolytic peptide map of the comigrating in vitro translation p ... | 1980 | 18631661 |
| translation of potyvirus rna in a rabbit reticulocyte lysate: cell-free translation strategy and a genetic map of the potyviral genome. | the translations of tobacco etch virus (tev) rna and pepper mottle virus (pemv) in a rabbit reticulocyte lysate were analyzed to determine the effect of rna quality on template activity and to identify all the products of the potyviral genome. the size distribution of the in vitro translation products, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), was dependent on rna quality. full-length rna stimulated the cell-free synthesis primarily of a 87,000 (87k) mo ... | 1980 | 18631662 |
| in vitro translation of tobacco etch virus rna. | the wheat-embryo-derived cell-free system was optimized for translation of tobacco etch virus (tev) rna. when examined by polyacrylamide gel electrophoresis, the product of the tev rna stimulated system proved to be a single protein with a molecular weight of 40,000. this finding is different from results obtained when tev rna is used as a template in the rabbit reticulocyte lysate in vitro protein-synthesizing system. | 1980 | 18631719 |
| the rna of tobacco etch virus: further characterization and detection of protein linked to rna. | tev-rna was separated into poly(a)+ and poly(a)- rna by affinity chromatography. both classes of rna have similar base ratios. the poly(a) segment in the rna was shown to be located at the 3'-oh end by dihydroxyborylaminoethyl-cellulose chromatography. although the poly(a) was heterodisperse in length, ranging from about 200 to less than 33 amp residues, two size classes averaging about 150 and 30 amp residues, respectively, could be recognized. the presence of a genome-linked protein of molecul ... | 1981 | 18635072 |
| cell-free translation of tobacco vein mottling virus rna. ii. immunoprecipitation of products by antisera to cylindrical inclusion, nuclear inclusion, and helper component proteins. | the genomic rna of tobacco vein mottling virus (tvmv) was translated in a cell-free system derived from rabbit reticulocytes. antisera against tvmv coat protein, tvmv cylindrical inclusions, the helper component required for aphid transmission of tvmv, and the 49- and 54-kd nuclear inclusion proteins of tobacco etch virus (tev) were used to characterize the translational products. each of the five antisera precipitated a distinctive pattern of polypeptides. specificity of immunoprecipitation was ... | 1983 | 18638869 |
| detection and cell-free translation of subgenomic rnas of tobacco etch virus. | rna extracted from purified tobacco etch virus and virus-infected tissue was examined by the northern hybridization technique and found to contain several subgenomic-sized viral rna species in addition to the 3 x 10(6) mw genomic rna. of the 10 subgenomic-sized rnas detected (2.03, 1.24, 1.14, 1.01, 0.84, 0.62, 0.55, 0.46, 0.34, and 0.28 x 10(6) mw), 4 were found in both virion rna and infected tissue rna, 3 others only in virion rna, and 3 only in infected tissue rna. of the last three, two may ... | 1983 | 18638889 |
| helper components of two potyviruses are serologically distinct. | the specificity of antisera to helper component (hc) from tobacco vein mottling virus (tvmv)- or potato virus y (pvy)-infected tobacco plants was tested in immunoprecipitation and immunoabsorption chromatography experiments. treatment with the homologous antiserum abolished or drastically reduced the activity of either tvmv-hc or pvy-hc, as measured by their ability to effect aphid transmission of purified tobacco etch virus, while the heterologous antiserum had little or no effect on hc activit ... | 1983 | 18638913 |
| analysis of viral rna isolated from tobacco leaf tissue infected with tobacco etch virus. | total rna has been isolated from tobacco seedlings systemically infected with the potyvirus, tobacco etch virus (tev). analysis of this rna by agarose gel electrophoresis under denaturing conditions, in conjunction with gel hybridization using various virus-specific nucleic acid probes, revealed genomic length tev rna and eight zones of less-than-full-length rna. however, reconstitution experiments demonstrated that the zones were electrophoretic artifacts and not authentic subgenomic rnas. furt ... | 1983 | 18639175 |
| translation of soybean mosaic virus rna in vitro: evidence of protein processing. | the genomic rna of soybean mosaic virus (smv), a member of the potyvirus group of plant viruses, was translated in both the wheat germ and reticulocyte cell free systems to identify some viral encoded proteins and as an approach to determining the translational strategy of the virus. the rna was translated into the same specific set of 10 to 12 polypeptides in both in vitro systems. immunological tests and peptide analyses indicate that six translation products are related to smv coat protein, a ... | 1984 | 18639801 |
| immunoprecipitation analysis of potyviral in vitro translation products using antisera to helper component of tobacco vein mottling virus and potato virus y. | the serological relationships of the products of in vitro translation of the rna of various potyviruses were analyzed by using antisera to helper component (hc) from tobacco plants infected with either tobacco vein mottling virus (tvmv) or potato virus y (pvy). the pvy-hc antiserum immunoprecipitated a specific pvy-rna translation product; this product was not reactive with antisera to pvy-induced cylindrical inclusion protein or capsid protein or to the two tobacco etch virus nuclear inclusion ... | 1984 | 18639815 |
| isolation and partial characterization of the amorphous cytoplasmic inclusions associated with infections caused by two potyviruses. | the potyviruses pepper mottle (pemv) and watermelon mosaic virus-1, a strain of papaya ringspot (prsv-w), induce the formation of a second type of cytoplasmic inclusion in addition to cylindrical (pinwheel) inclusions. the conspicuous aggregates of electron-dense material with imperfect spherical shapes have been called amorphous inclusions (ai). the ai were isolated from extracts of infected tissue using a combination of clarification with triton x-100 and low-speed centrifugations through sucr ... | 1985 | 18639842 |
| topographic analysis of tobacco etch virus capsid protein epitopes. | monoclonal antibodies have been prepared, which react with capsid protein of an aphid-transmitted isolate of tobacco etch virus (tev). ten different monoclonal antibodies were characterized with reference to (1) antibody class, (2) reactivity with different plant virus antigens, (3) the spatial relationship between epitopes, and (4) whether these epitopes were located on the exterior surface of the virion. three monoclonal antibodies were specific for tev isolates. these monoclonal antibodies re ... | 1985 | 18640514 |
| biochemical analysis of the capsid protein gene and capsid protein of tobacco etch virus: n-terminal amino acids are located on the virion's surface. | the sequence of the 1491 nucleotides found at the 3' end of the genome of the highly aphid-transmissible (hat) isolate of tobacco etch virus (tev) has been determined. the nucleotide sequence of the capsid protein gene has been identified and compared with the corresponding region of the not-aphid-transmissible (nat) isolate of tev and with pepper mottle virus (pemv). the deduced amino acid sequences of the two tev capsid proteins displayed 98% homology and a 66% homology with pemv capsid protei ... | 1985 | 18640560 |
| construction of a recombinant vaccinia virus which expresses immunoreactive plant virus proteins. | a chimeric transcription unit was assembled consisting of a vaccinia virus promoter linked to a 2400 bp(3) double-stranded complementary dna (cdna) made to the 3' end of the genomic rna of the plant pathogen, tobacco etch virus (tev). marker rescue techniques were used to introduce virus recombinant genes into the vaccinia virus genome. the recombinant virus (designated wnat) was isolated, purified, and subjected to molecular genetic analyses. the ability of wnat to direct the expression of plan ... | 1986 | 18640593 |
| coat protein of potyviruses. 2. amino acid sequence of the coat protein of potato virus y. | the amino acid sequence of the coat protein of potato virus y (pvy), the type member of the potyvirus group, has been determined by protein sequencing techniques. the protein contains 267 amino acid residues with a calculated mol wt of 29,945. a comparison of the pvy coat protein sequence with those of tobacco etch virus (tev) and pepper mottle virus (pemv) predicted from nucleotide sequence data (r. f. allison, j. g. sorenson, m. e. kelly, f. b. armstrong, and w. g. dougherty, proc. natl. acad. ... | 1986 | 18640638 |
| the effect of helper component on the uptake and localization of potyviruses in myzus persicae. | 125i-labeled virions were used to determine whether helper component (hc) affected the uptake or distribution of potyviruses in aphids. aphids were allowed to acquire purified, 125i-labeled tobacco etch virus or potato virus y mixed with hc or with inactivated hc. helper component had no effect upon uptake of labeled virus, as measured by gamma counting. autoradiography of freeze-sectioned aphids revealed, however, that in the presence of hc, label was associated with the maxillary stylets and w ... | 1986 | 18640647 |
| the nucleotide sequence of the coding region of tobacco etch virus genomic rna: evidence for the synthesis of a single polyprotein. | the complete nucleotide sequence of the tobacco etch virus (tev) rna genome has been determined excepting only the nucleotide(s) present at the extreme 5' terminus. the assembled tev genomic sequence is 9496 nucleotides in length followed by a polyadenylated tract ranging from 20 to 140 residues. a computer search of the sequence reveals the following. a 5' untranslated region, rich in adenosine and uridine, is present between nucleotides 1 and 144. a putative initiation codon, at nucleotides 14 ... | 1986 | 18640649 |
| potyviral proteins share amino acid sequence homology with picorna-, como-, and caulimoviral proteins. | the predicted amino acid sequences of the polyproteins of two potyviruses, tobacco vein mottling virus (tvmv) and tobacco etch virus (tev), were compared to each other, to proteins of other viruses, and to the national biomedical research foundation protein sequence bank. three potyviral proteins, the cylindrical inclusion and the two nuclear inclusion proteins, were found to be homologous to proteins considered to be involved in the replication and expression of picorna- and comovirus rna. a lo ... | 1987 | 18644561 |
| processing of the tobacco etch virus 49k protease requires autoproteolysis. | the final products encoded by the tobacco etch virus genome arise by proteolytic cleavage of a single large polyprotein precursor. processing of the polyprotein at several sites requires the activity of a viral protease of 49,000 molecular weight (49k). we have examined the excision of the 49k protease from polyproteins translated from defined rna transcripts. polyproteins containing an intact 49k protein were efficiently processed after synthesis in a rabbit reticulocyte lysate to yield the 49k ... | 1987 | 18644573 |
| exploring the activity of tobacco etch virus protease in detergent solutions. | tobacco etch virus (tev) protease is generally used to remove affinity tags from target proteins. it has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. the aim of this study was to explore and evaluate this further. hence, affinity tag removal with tev protease was tested from three membrane proteins (a pgp synthase and two cora homologs) in the presence of 16 different detergents commo ... | 2008 | 18682245 |
| microbial bio-production of a recombinant stimuli-responsive biosurfactant. | biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. the production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of impr ... | 2009 | 18683262 |
| changes in the gene expression profile of arabidopsis thaliana after infection with tobacco etch virus. | tobacco etch potyvirus (tev) has been extensively used as model system for the study of positive-sense rna virus infecting plants. tev ability to infect arabidopsis thaliana varies among ecotypes. in this study, changes in gene expression of a. thaliana ecotype ler infected with tev have been explored using long-oligonucleotide arrays. a. thaliana ler is a susceptible host that allows systemic movement, although the viral load is low and syndrome induced ranges from asymptomatic to mild. gene ex ... | 2008 | 18684336 |
| effects of poly(a)-binding protein on the interactions of translation initiation factor eif4f and eif4f.4b with internal ribosome entry site (ires) of tobacco etch virus rna. | in wheat germ, the interaction between poly(a)-binding protein and eukaryotic initiation factor eif 4g increases the affinity of eif4e for the cap by 20-40-fold. recent findings that wheat germ eif4g is required for interaction with the ires, pseudoknot 1 (pk1), of tobacco etch virus to promote cap-independent translation led us to investigate the effects of pabp on the interaction of eif4f with pk1. the fluorescence anisotropy data showed addition of pabp to eif4f increased the binding affinity ... | 2008 | 18692164 |
| the pleiotropic cost of host-specialization in tobacco etch potyvirus. | host-range expansion is thought to allow viruses to broaden their ecological niches by allowing access to new resources. however, traits governing the infection of multiple hosts may decrease fitness in the original one due to the pleiotropic effect of adaptive mutations that may give rise to fitness tradeoffs across hosts. here, we have experimentally examined the consequences of host-specialization in the plant pathogen tobacco etch potyvirus (tev). replicate populations of tev were allowed to ... | 2008 | 18765302 |
| from hypo- to hypersuppression: effect of amino acid substitutions on the rna-silencing suppressor activity of the tobacco etch potyvirus hc-pro. | rna silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. most plant viruses have evolved proteins that suppress rna silencing and that in many cases are multifunctional. tobacco etch potyvirus (tev) hc-pro protein suppresses rna silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. in this study, we have generated 28 hc-pro amino acid su ... | 2008 | 18780745 |
| internal core protein cleavage leaves the hepatitis b virus capsid intact and enhances its capacity for surface display of heterologous whole chain proteins. | virus capsids find increasing use as nanoparticulate platforms for the surface display of heterologous ligands, including as multivalent vaccine carriers. presentation on the icosahedral hepatitis b virus capsid (hbcag) is known to strongly enhance immunogenicity of foreign sequences, most efficiently if they are inserted into the dominant c/e1 b cell epitope, a surface-exposed loop in the center of the constituent core protein primary sequence. even some complete proteins were successfully inse ... | 2008 | 18826949 |
| a simple and efficient expression and purification system using two newly constructed vectors. | structural biology places a high demand on proteins both in terms of quality and quantity. although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. here, we report a new system which combines improved expression, solubility screening and purification efficiency. the system is based on two newly constructed vectors, pehistev and pehisgfptev derived from a pet vector. both vectors generate a construct wi ... | 2009 | 18845260 |
| preparation and characterization of a novel recombinant human parathyroid hormone (1-34) analog (gly1-gln26-rhpth(1-34)) with enhanced biological activity. | a recombinant human parathyroid hormone (rhpth) fragment (gly1-gln26-rhpth(1-34)) which contains two amino acids substitutions (gly1 and gln26) was acquired through escherichia coli expression system using a soluble fusion protein strategy. the soluble fusion protein mbp-gly1-gln26-rhpth(1-34) was harvested after purification by phenyl-sepharose f.f and q-sepharose f.f chromatographies. following tobacco etch virus (tev) protease cleavage and further purification by sp-sepharose f.f chromatograp ... | 2008 | 18855760 |
| covalent immobilization of tobacco-etch-virus nia protease: a useful tool for cleavage of the histidine tag of recombinant proteins. | addition of tags [such as his (histidine) tags] is extremely helpful for the affinity purification of recombinant proteins. in several cases, these tags must be removed before performing functional and structural studies. the enzyme most frequently used to cleave tags of recombinant proteins is the tev-protease (tobacco-etch-virus nia protease). the continuous production of this enzyme in soluble form is quite an expensive process and not easily accessible to many laboratories. thus an interesti ... | 2009 | 18937642 |
| host range and characterization of sunflower mosaic virus. | abstract sunflower mosaic is caused by a putative member of the family potyviridae. sunflower mosaic virus (sumv) was characterized in terms of host range, physical and biological characteristics, and partial nucleotide and amino acid sequence. cells infected with sumv had cytoplasmic inclusion bodies typical of potyviruses. of 74 genera tested, only species in helianthus, sanvitalia, and zinnia, all asteraceae, were systemic hosts. commercial sunflower hybrids from the united states, europe, an ... | 2002 | 18943264 |
| rna-protein crosslink mapping using tev protease. | characterization of novel rna-protein interactions often demands physical mapping of the rna binding sites in the protein. this can sometimes be accomplished using radioactively labeled rna in covalent rna-protein crosslinking experiments. the position of the radioactive label crosslinked to the protein can then be determined by fragmentation of the protein using a battery of sequence-specific proteolytic enzymes or chemical reagents. however, there are typically many cleavage sites in the natur ... | 2008 | 18982293 |
| screening of fusion partners for high yield expression and purification of bioactive viscotoxins. | viscotoxins are small cationic proteins found in european mistletoe viscum album. they are highly toxic towards phytopathogenic fungi and cancer cells. heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. here, we evaluated 13 different proteins as a fusion partners for expression in escherichia coli cells: his6 tag and his6-tagged versions of gb1, zz tag, ... | 2009 | 18983922 |
| hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in escherichia coli. | insolubility of recombinant proteins in escherichia coli is a major impediment to their production for structural and functional studies. one way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. e. coli maltose-binding protein (mbp) has emerged as one of the most effective solubilizing agents. in this chapter, we describe how to construct combinatorially-tagged his(6)mbp fusion proteins by recombinational cloning and how to evaluate their yield and ... | 2009 | 18988025 |
| expression and purification of soluble his(6)-tagged tev protease. | this chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (tev) protease in escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. the protease is initially produced as a fusion to the c-terminus of e. coli maltose binding protein (mbp), which causes it to accumulate in a soluble and active form rather than in inclusion bodies. the fusion pr ... | 2009 | 18988033 |
| molecular cloning, overproduction, purification and biochemical characterization of the p39 nsp2 protease domains encoded by three alphaviruses. | alphaviruses cause serious diseases that pose a potential health threat to both humans and livestock. the nonstructural protein 2 (nsp2) encoded by alphaviruses is a multifunctional enzyme that is essential for viral replication and maturation. its 39-kda c-terminal domain (nsp2pro) is a cysteine protease that is responsible for cleaving a viral polyprotein at three sites to generate nonstructural proteins 1, 2, 3 and 4. in the present study, we evaluated nsp2pro domains from the following three ... | 2009 | 19013248 |
| the application of tandem-affinity purification to candida albicans. | tandem-affinity purification (tap) tagging systems, developed by the research group of bertrand seraphin and others, are a means of isolating physiologically relevant protein and protein-nucleic acid complexes. where complete (or nearly complete) genome sequence data are available for the organism from which the complexes are isolated, their components can be readily identified using mass spectrometry. the most widely used tap-tag consists of a proximal calmodulin-binding peptide (cbp) and a dis ... | 2009 | 19152045 |
| efficient cleavage of problematic tobacco etch virus (tev)-protein arginine methyltransferase constructs. | protein arginine methyltransferases (prmts) are enzymes that are involved in many biological processes. several studies have shown that the identity of the n-terminal fusion tag affects the substrate selectivity of prmts. therefore, to accurately study substrate recognition, it is imperative that a tagless prmt be used. however, cleavage of tagged prmts has been problematic. we have developed a successful method by which untagged prmts can be made using a tobacco etch virus (tev) cleavage site a ... | 2009 | 19167339 |
| quantitative evaluation of six different viral suppressors of silencing using image analysis of transient gfp expression. | the effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of gfp expression. suppressors were introduced into lima bean cotyledonary tissues either as 3'-gfp translational fusions or as co-introductions with the gfp gene on a separate plasmid. the resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introd ... | 2009 | 19198843 |
| virus diseases in the tobacco fields of guilan and western azerbaijan provinces of iran. | tobacco (nicotiana tabacum l.) is one of the important industrial plants in iran. viruses as an important group of plant pathogens cause many losses on the quality and quantity of tobacco crop. there was few information on the types of plant viruses infecting the tobacco fields of guilan and almost no information for western azerbaijan province. during 2005-2007, leaf samples were taken from symptomatic plants in the growing areas of these two provinces. the observed symptoms on plants in the fi ... | 2008 | 19226768 |
| crystal structure of a novel dimeric form of ns5a domain i protein from hepatitis c virus. | a new protein expression vector design utilizing an n-terminal six-histidine tag and tobacco etch virus protease cleavage site upstream of the hepatitis c virus ns5a sequence has resulted in a more straightforward purification method and improved yields of purified ns5a domain i protein. high-resolution diffracting crystals of ns5a domain i (amino acids 33 to 202) [ns5a(33-202)] were obtained by using detergent additive crystallization screens, leading to the structure of a homodimer which is or ... | 2009 | 19244328 |
| purification of recombinant apolipoproteins a-i and a-iv and efficient affinity tag cleavage by tobacco etch virus protease. | the expression of recombinant apolipoproteins provides experimental avenues that are not possible with plasma purified protein. the ability to specifically mutate residues or delete entire regions has proven to be a valuable tool for understanding the structure and function of apolipoproteins. a common feature of many recombinant systems is an affinity tag that allows for straightforward and high-yield purification of the target protein. a specific protease can then cleave the tag and yield the ... | 2009 | 19318686 |
| n-terminal of papaya ringspot virus type-w (prsv-w) helper component proteinase (hc-pro) is essential for prsv systemic infection in zucchini. | the papaya ringspot virus (prsv) is one of the limiting factors affecting papaya and cucurbits production worldwide. prsv belongs to the potyvirus genus which consists of 30% of known plant viruses. two serological closely related strains, namely type-p and -w, have been reported. prsv type-p infects both papaya and cucurbits, while type-w infects only cucurbits. the genome of prsv thailand isolate consists of a (+) rna molecule of 10323 nucleotides, which is first translated into a single polyp ... | 2009 | 19322647 |
| upper-limit mutation rate estimation for a plant rna virus. | it is generally accepted that mutation rates of rna viruses are inherently high due to the lack of proofreading mechanisms. however, direct estimates of mutation rate are surprisingly scarce, in particular for plant viruses. here, based on the analysis of in vivo mutation frequencies in tobacco etch virus, we calculate an upper-bound mutation rate estimation of 3x10(-5) per site and per round of replication; a value which turns out to be undistinguishable from the methodological error. nonethele ... | 2009 | 19324646 |
| fret-based optical assay for monitoring riboswitch activation. | riboswitches are regulatory rnas located in the 5'-untranslated region of mrna sequences that recognize and bind to small molecules and regulate the expression of downstream genes. creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. in our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (fret) between two genetically encoded fluorescent pro ... | 2009 | 19358526 |
| efficient protein depletion by genetically controlled deprotection of a dormant n-degron. | methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. we developed a dormant n-degron that can be attached to the n-terminus of a protein of interest. upon expression of a site-specific protease, the dormant n-degron becomes deprotected. the n-degron then targets itself and the attached protein for rapid proteasomal de ... | 2009 | 19401679 |
| synthesis of polymerizable protein monomers for protein-acrylamide hydrogel formation. | a novel method to produce protein polymer conjugates for protein-acrylamide hydrogel formation is described. alkenes are incorporated onto the n-terminus of expressed proteins to produce polymerizable protein monomers that can be utilized in protein-acrylamide copolymerization. a 4-vinylbenzoic acid thioester was synthesized and attached to the n-termini of two protein models, the immunoglobulin-binding protein protein g and the bacterial enzyme xanthine-guanine phosphoribosyltransferase (gprt), ... | 2009 | 19453166 |
| discovery of spiro-piperidine inhibitors and their modulation of the dynamics of the m2 proton channel from influenza a virus. | amantadine has been used for decades as an inhibitor of the influenza a virus m2 protein (am2) in the prophylaxis and treatment of influenza a infections, but its clinical use has been limited by its central nervous system (cns) side effects as well as emerging drug-resistant strains of the virus. with the goal of searching for new classes of m2 inhibitors, a structure-activity relation study based on 2-[3-azaspiro(5,5)undecanol]-2-imidazoline (bl-1743) was initiated. the first generation bl-174 ... | 2009 | 19469531 |
| systematic characterization of the protein interaction network and protein complexes in saccharomyces cerevisiae using tandem affinity purification and mass spectrometry. | defining protein complexes is a vital aspect of cell biology because cellular processes are often carried out by stable protein complexes and their characterization often provides insights into their function. accurate identification of the interacting proteins in macromolecular complexes is easiest after purification to near homogeneity. to this end, the tandem affinity purification (tap) system with subsequent protein identification by high-throughput mass spectrometry was developed (1, 2) to ... | 2009 | 19521826 |
| the chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly. | one of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. here we report a chromatography-free isolation and purification process for recombinant peptide expressed in escherichia coli (e. coli). initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the e. coli homogenate. release is followed by selective solvent precipit ... | 2009 | 19530081 |
| sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from escherichia coli. | biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. to this end, a se ... | 2009 | 19544035 |
| expression and purification of recombinant arginine decarboxylase (spea) from escherichia coli. | the crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase's structure is not resolved yet. in order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (adc; ec 4.1.1.19, encoded by the spea gene) from escherichia coli in the t7 expression system as a cleavable poly-his-tagged fusion construct. the expressed recombinant his(10)-adc (77.3 kda) was first purified by ni-nta affini ... | 2010 | 19603287 |
| the determinant of potyvirus ability to overcome the rtm resistance of arabidopsis thaliana maps to the n-terminal region of the coat protein. | in arabidopsis thaliana columbia (col-0) plants, the restriction of tobacco etch virus (tev) long-distance movement involves at least three dominant rtm (restricted tev movement) genes named rtm1, rtm2, and rtm3. previous work has established that, while the rtm-mediated resistance is also effective against other potyviruses, such as plum pox virus (ppv) and lettuce mosaic virus (lmv), some isolates of these viruses are able to overcome the rtm mechanism. in order to identify the viral determina ... | 2009 | 19737103 |
| design of high-affinity s100-target hybrid proteins. | s100b and s100a10 are dimeric, ef-hand proteins. s100b undergoes a calcium-dependent conformational change allowing it to interact with a short contiguous sequence from the actin-capping protein capz (trtk12). s100a10 does not bind calcium but is able to recruit the n-terminus of annexin a2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins trpv5/6. in this work, we have designed, expressed, purified, and characterized tw ... | 2009 | 19827097 |
| rapid modification of proteins using a rapamycin-inducible tobacco etch virus protease system. | the ability to disrupt the function of a specific protein on a rapid time scale provides a powerful tool for biomedical research. specific proteases provide a potential method to selectively cleave a chosen protein, but rapid control of protease activity is difficult. | 2009 | 19830250 |
| kinetic mechanism for the binding of eif4f and tobacco etch virus internal ribosome entry site rna: effects of eif4b and poly(a)-binding protein. | the wheat germ eukaryotic translation initiation factor (eif) 4f binds tightly to the mrna internal ribosome entry site (ires) of tobacco etch virus (tev) to promote translation initiation. when eif4f is limiting, tev is preferentially translated compared with host cell mrna. to gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eif4f binding to tev ires were examined. the association rate constant (k(on)) and dissociation rate con ... | 2009 | 19858189 |
| compensatory molecular evolution of hc-pro, an rna-silencing suppressor from a plant rna virus. | rna silencing is a eukaryotic mechanism involved in several cellular processes, one example being a sequence-specific antiviral defense. many plant viruses have developed counterdefensive proteins that in many instances are multifunctional, such as helper component protease (hc-pro) of tobacco etch virus (tev). in a previous work, a collection of mutants with amino acid replacements in tev hc-pro was generated, and their effects in the capacity of suppressing rna silencing were quantified in a t ... | 2010 | 19906792 |
| the tobacco etch virus p3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments. | plant potyviruses encode two membrane proteins, 6k and p3. the 6k protein has been shown to induce virus replication vesicles. however, the function of p3 remains unclear. in this study, subcellular localization of the tobacco etch virus (tev) p3 protein was investigated in nicotiana benthamiana leaf cells. the tev p3 protein localized on the endoplasmic reticulum (er) membrane and formed punctate inclusions in association with the golgi apparatus. the trafficking of p3 to the golgi was mediated ... | 2010 | 19945728 |
| integrated analysis of receptor activation and downstream signaling with extassays. | the ability to measure multiple cellular signaling events is essential to better understand the underlying complex biological processes that occur in living cells. microarray-based technologies are now commonly used to study changes in transcription. this information, however, is not sufficient to understand the regulatory mechanisms that lead to gene expression changes. here we present an approach to monitor signaling events upstream of gene expression. we coupled different reporter gene assays ... | 2010 | 20010833 |
| properties of a homogeneous c-lobe prepared by introduction of a tev cleavage site between the lobes of human transferrin. | essential to iron transport and delivery, human serum transferrin (htf) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the n- and c-lobes). a complete description of iron release from htf, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. although production of large amounts of isolated n-lobe and full-length htf has been well documented, attempts to produce the c-lobe (by recombi ... | 2010 | 20064616 |
| hc-pro hypo- and hypersuppressor mutants: differences in viral sirna accumulation in vivo and sirna binding activity in vitro. | viruses have evolved mechanisms to suppress the rna silencing defense of their hosts, allowing replication and systemic colonization. in a recent study, we found that the effect of mutations in the rna silencing suppressor of tobacco etch virus (tev) was variable, ranging from complete abolition of suppressor activity to significantly stronger suppression. whereas hyposuppressor mutants were less virulent and accumulated fewer viral particles than the wild type, hypersuppressors induced symptoms ... | 2010 | 20091191 |
| evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics. | protease cleavage site recognition motifs can be identified using protease substrate discovery methodologies, but typically exhibit non-optimal specificity and activity. to enable evolutionary optimization of substrate cleavage kinetics, a two-color cellular library of peptide substrates (clips) methodology was developed. two-color clips was applied to identify peptide substrates for the tobacco etch virus (tev) protease from a random pentapeptide library, which were then optimized by screening ... | 2010 | 20148412 |
| stability of tobacco etch virus infectious clones in plasmid vectors. | tobacco etch virus (tev) has been traditionally used as a model to research many aspects of the molecular biology of plant rna virus and, more recently, experimental evolution. however, the only plasmid of this virus species with an infectious clone that has been commonly available to research (ptev7da) is rather unstable when propagated in the bacterium escherichia coli. here, the tev infectious clone contained in ptev7da is used to construct three new plasmids that allowed infecting the host p ... | 2010 | 20152868 |
| a novel method for high-level production of tev protease by superfolder gfp tag. | because of its stringent sequence specificity, tobacco etch virus (tev) protease is widely used to remove fusion tags from recombinant proteins. due to the poor solubility of tev protease, many strategies have been employed to increase the expression level of this enzyme. in our work, we introduced a novel method to produce tev protease by using visible superfolder green fluorescent protein (sfgfp) as the fusion tag. the soluble production and catalytic activity of six variants of sfgfp-tev was ... | 2010 | 20182554 |
| identification of novel interacting protein partners of smn using tandem affinity purification. | mutations in the survival motor neuron (smn) gene cause spinal muscular atrophy (sma), a neuromuscular disease associated with muscle weakness that progresses to paralysis, respiratory distress, and ultimately death. both neurons and muscle are severely affected in this disease. tandem affinity purification (tap) has emerged as a useful tool for studying protein complexes in vitro. we have used this purification system to discover novel smn interacting partners in c2c12 muscle and pc12 neuronal ... | 2010 | 20201562 |
| purification, crystallization and preliminary data analysis of focb, a transcription factor regulating fimbrial adhesin expression in uropathogenic escherichia coli. | the transcription factor focb belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic escherichia coli. recent findings suggest that focb-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. however, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. the 109-amino-acid focb transcrip ... | 2010 | 20208176 |
| expression and purification of ataxin-1 protein. | ataxin-1 is part of a larger family of polyglutamine-containing proteins that is linked to nine distinct neurodegenerative disorders. there are no known effective therapies for any of these expanded polyglutamine tract disorders. one possible reason for this is the lack of sufficient amounts of pure polyglutamine-containing proteins suitable for biochemical and conformational studies. here, we show that we were able to successfully purify a non-pathological, wild-type human ataxin-1 protein cont ... | 2010 | 20304006 |
| inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation. | caspase-8 is a cysteine protease activated by membrane-bound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be re ... | 2010 | 20308068 |
| tomato chocolate spot virus, a member of a new torradovirus species that causes a necrosis-associated disease of tomato in guatemala. | tomatoes in guatemala have been affected by a new disease, locally known as "mancha de chocolate" (chocolate spot). the disease is characterized by distinct necrotic spots on leaves, stems and petioles that eventually expand and cause a dieback of apical tissues. samples from symptomatic plants tested negative for infection by tomato spotted wilt virus, tobacco streak virus, tobacco etch virus and other known tomato-infecting viruses. a virus-like agent was sap-transmitted from diseased tissue t ... | 2010 | 20376682 |
| application of "homogeneous assay for fluorescence concentrated on membrane" to the analysis of the substrate specificity of protease. | the utility of the homogeneous assay for fluorescence concentrated on membrane (hafcom) in the analysis of the substrate specificity of protease was investigated using tobacco etch virus (tev) protease. the v(max) of tev protease against variants of a substrate was obtained by a simple procedure. it was considered that hafcom was more accurate than other endpoint measurements of protease assay. | 2010 | 20378974 |
| production of thymosin alpha1 via non-enzymatic acetylation of the recombinant precursor. | human thymosin alpha1 is an effective immune system enhancer for the treatment of cancer and viral diseases. therefore the development of new methods for its synthesis is an urgent problem. in the present work, we propose an efficient scalable scheme for the production of recombinant thymosin alpha1. we used an expression system based on the pet32b+ plasmid and escherichia coli strain er2566 to obtain a fusion protein consisting of thymosin alpha1 and thioredoxin separated by a tev (tobacco etch ... | 2010 | 20408810 |
| the rate and spectrum of spontaneous mutations in a plant rna virus. | knowing mutation rates and the molecular spectrum of spontaneous mutations is important to understanding how the genetic composition of viral populations evolves. previous studies have shown that the rate of spontaneous mutations for rna viruses widely varies between 0.01 and 2 mutations per genome and generation, with plant rna viruses always occupying the lower side of this range. however, this peculiarity of plant rna viruses is based on a very limited number of studies. here we analyze the s ... | 2010 | 20439778 |
| adaptation of tobacco etch potyvirus to a susceptible ecotype of arabidopsis thaliana capacitates it for systemic infection of resistant ecotypes. | viral pathogens continue to emerge among humans, domesticated animals and cultivated crops. the existence of genetic variance for resistance in the host population is crucial to the spread of an emerging virus. models predict that rapid spread decreases with the frequency and diversity of resistance alleles in the host population. however, empirical tests of this hypothesis are scarce. arabiodpsis thaliana--tobacco etch potyvirus (tev) provides an experimentally suitable pathosystem to explore t ... | 2010 | 20478894 |
| host-dependent differences during synergistic infection by potyviruses with potato virus x. | summary a comparative analysis of the synergistic interaction between pvx and either pvy or tev potyviruses was performed in nicotiana benthamiana and n. tabacum plants. in each pvx/potyvirus combination, doubly infected plants developed much more severe symptoms than singly infected ones. however, while pvx accumulation increased in doubly infected n. tabacum plants compared with singly infected plants, the accumulation of pvx did not vary drastically in doubly infected n. benthamiana plants wi ... | 2004 | 20565579 |
| overview and analysis of the polyprotein cleavage sites in the family potyviridae. | summary the genomes of plant viruses in the family potyviridae encode large polyproteins that are cut by virus-encoded proteases into ten mature proteins. three different types of protease have been identified, each of which cuts at sites with a distinctive sequence pattern. the experimental evidence for this specificity is reviewed and the cleavage site patterns are compiled for all sequenced species within the family. seven of the nine cleavage sites in each species are cut by the viral nia-pr ... | 2005 | 20565672 |
| the helper-component protease transmission factor of tobacco etch potyvirus binds specifically to an aphid ribosomal protein homologous to the laminin receptor precursor. | potyviruses are plant pathogens transmitted by aphids in a non-persistent manner. during transmission, the virus-encoded factor helper-component protease (hcpro) is presumed to act as a molecular bridge, mediating the reversible retention of virions to uncharacterized binding sites in the vector mouthparts. whilst the predicted interaction between hcpro and the coat protein (cp) of virions has been confirmed experimentally, the characterization of putative hcpro-specific receptors in aphids has ... | 2010 | 20631085 |
| interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry. | most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. advances in ms have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the tap (tandem affinity ... | 2010 | 20658971 |
| ring structure amino acids affect the suppressor activity of melon aphid-borne yellows virus p0 protein. | melon aphid-borne yellows virus (mabyv) is a newly identified polerovirus occurring in china. here, we demonstrate that the mabyv encoded p0 (p0(ma)) protein is a strong suppressor of post-transcriptional gene silencing (ptgs) with activity comparable to tobacco etch virus (tev) hc-pro. in addition we have shown that the lp f-box motif present at the n-terminus of p0(ma) is required for suppressor activity. detailed mutational analyses on p0(ma) revealed that changing the conserved trp 212 with ... | 2010 | 20667575 |
| photocatalytic activity of protein-conjugated cds nanoparticles. | colloidal cds nanoparticles are conjugated with a variety of proteins, including enhanced yellow fluorescent protein, tobacco etch virus protease (tev), lysozyme, and bacterial cytochrome p450 cyp152a1, and the photochemical properties of the resulting conjugates are analyzed by epr spectroscopy and hydroxyl radical-specific fluorimetric assay. while irradiation of bare cds colloids leads to photogeneration of hydroxyl and superoxide radicals, it is surprisingly observed that coating of the cds ... | 2010 | 20721950 |
| poly(a) tail affects equilibrium and thermodynamic behavior of tobacco etch virus mrna with translation initiation factors eif4f, eif4b and pabp. | we have investigated the effects of poly(a)-tail on binding of eif4f, eif4b and pabp with tobacco etch virus (tev) ires rna. the fluorescence anisotropy data showed that the addition of poly(a)(20) increases the binding affinity of eif4f·4b and eif4f·pabp complexes to ires rna ~2- and 4-fold, respectively. however, the binding affinity of eif4f with pk1 was enhanced ~11-fold with the addition of pabp, eif4b, and poly(a)(20) together. whereas, poly(a)(20) alone increases the binding affinity of e ... | 2010 | 20723624 |
| activation of specific apoptotic caspases with an engineered small-molecule-activated protease. | apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. to dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. our approach uses a split-tobacco etch virus (tev) protease under small-molecule control, which we call the sniper, with caspase alleles containing genetically encoded tev cleavage site ... | 2010 | 20723762 |
| a new tagged-tev protease: construction, optimisation of production, purification and test activity. | the tobacco etch virus (tev) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity. in this work, we present a new recombinant form of tev termed streptag ii-tev for high-level production and purification of tev protease from escherichia coli and compare it to the hexahistidine (6xhis) tagged version of tev. the effects of varying the host strain, the bacterial induction temperature (25, 30 and 37°c) and the iptg inducer concent ... | 2011 | 20817099 |
| novel fluorescence-assisted whole-cell assay for engineering and characterization of proteases and their substrates. | we have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. the method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the escherichia coli cytoplasm. we demonstrated that this system can reveal differenc ... | 2010 | 20851955 |
| structural determinants of tobacco vein mottling virus protease substrate specificity. | tobacco vein mottling virus (tvmv) is a member of the potyviridae, one of the largest families of plant viruses. the tvmv genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. seven of the nine cleavage events are carried out by the nia protease. its homolog from the tobacco etch virus (tev) is a widely used reagent for the removal of affinity tags from recombinant proteins. although tvmv protease is a close relative of tev protea ... | 2010 | 20862670 |
| the puri family of expression vectors: a versatile set of ligation independent cloning plasmids for producing recombinant his-fusion proteins. | a family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant his-tagged fusion proteins in escherichia coli. these are based on puri2 and puri3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. the newly designed vectors combines two different promoters (lpp(p)-5 and t7 rna polymerase ø10), two different endoprotease recognition sites for the his₆-tag removal (e ... | 2010 | 21055470 |
| tissue blot immunoassay and direct rt-pcr of cucumoviruses and potyviruses from the same nitropure nitrocellulose membrane. | a method is described for using nitropure nitrocellulose (npn) membranes as an effective solid matrix for retrieval of template rna of three potyviruses, tobacco etch virus, soybean mosaic virus and turnip mosaic virus, and two cucumoviruses, cucumber mosaic virus and peanut stunt virus. these npn membranes were also used for tissue blot immunosorbent assays (tbias) to identify and detect plant viruses. for rna detection, discs from dried membranes blotted with infected tissue were minimally cle ... | 2010 | 21126542 |
| targeted protein depletion in saccharomyces cerevisiae by activation of a bidirectional degron. | tools for in vivo manipulation of protein abundance or activity are highly beneficial for life science research. protein stability can be efficiently controlled by conditional degrons, which induce target protein degradation at restrictive conditions. | 2010 | 21190544 |
| substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay. | site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (tevp). the ... | 2011 | 21267463 |
| studying g protein-coupled receptor activation using split-tobacco etch virus assays. | g protein-coupled receptors (gpcrs) constitute the largest receptor family in mammals and represent important drug targets. signaling through gpcrs mediates physiological effects that are strongly dependent on the cellular context. therefore, the availability of assays monitoring gpcr activation applicable in different cell types could help to better understand gpcr functions and to realize the potential of known substances as well as novel ones. here we introduce a split-tev (tobacco etch virus ... | 2011 | 21295005 |
| virus infection suppresses nicotiana benthamiana adaptive phenotypic plasticity. | competition and parasitism are two important selective forces that shape life-histories, migration rates and population dynamics. recently, it has been shown in various pathosystems that parasites can modify intraspecific competition, thus generating an indirect cost of parasitism. here, we investigated if this phenomenon was present in a plant-potyvirus system using two viruses of different virulence (tobacco etch virus and turnip mosaic virus). moreover, we asked if parasitism interacted with ... | 2011 | 21359142 |
| application of tev protease in protein production. | in many cases, the analysis of a specific protein is impeded by the inability to purify large amounts of it from a native source. proteins of interest may be present in minute quantities and/or purification may be plagued with technical problems. recombinant dna methodologies have enabled researchers to circumvent some of these limitations by producing and purifying large quantities of protein in a nonnative system. various systems and strategies have been successfully employed, depending on the ... | 1998 | 21390844 |
| effect of lead (pb) on the systemic movement of rna viruses in tobacco (nicotiana tabacum var. turkish). | effect of various lead (pb) concentrations on the systemic movement of rna viruses was examined in tobacco plants. prior to inoculation, plants were grown hydroponically for 6 days in hoagland's solution supplemented with five concentrations of lead nitrate [pb(no(3))(2)]: 0.0 (control), 10, 15, 50, and 100 µm. four different rna viruses with different cell-to-cell movement mechanisms were used. two weeks after inoculation lower and upper leaves of each treatment were harvested and examined for ... | 2011 | 21404008 |
| predictive mutagenesis of ligation-independent cloning (lic) vectors for protein expression and site-specific chemical conjugation. | ligation-independent cloning (lic) allows for cloning of dna constructs independent of insert restriction sites and ligases. however, any required mutations are typically introduced by additional, time-consuming steps. we present a rapid, inexpensive method for mutagenesis in the 5' lic site of expression constructs and report on the construction of expression vectors with n-terminal serine, cysteine, threonine, or tyrosine residues after tobacco etch virus (tev) protease cleavage. in a practica ... | 2011 | 21414287 |
| differences in accumulation and virulence determine the outcome of competition during tobacco etch virus coinfection. | understanding the evolution of virulence for rna viruses is essential for developing appropriate control strategies. although it has been usually assumed that virulence is a consequence of within-host replication of the parasite, viral strains may be highly virulent without experiencing large accumulation as a consequence of immunopathological host responses. using two strains of tobacco etch potyvirus (tev) that show a negative relationship between virulence and accumulation rate, we first expl ... | 2011 | 21423618 |
| a "two-hit" hypothesis for inclusion formation by carboxyl-terminal fragments of tdp-43 protein linked to rna depletion and impaired microtubule-dependent transport. | carboxyl-terminal fragments (ctfs) of tdp-43 aggregate to form the diagnostic signature inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the biological significance of these ctfs and how they are generated remain enigmatic. to address these issues, we engineered mammalian cells with an inducible tobacco etch virus (tev) protease that cleaves tdp-43 containing a tev cleavage site. regions of tdp-43 flanking the second rna recognition motif (rrm2) are efficien ... | 2011 | 21454607 |
| exploring the size limit of templates for inhibitors of the m2 ion channel of influenza a virus. | amantadine inhibits the m2 proton channel of influenza a virus, yet its clinical use has been limited by the rapid emergence of amantadine-resistant virus strains. we have synthesized and characterized a series of polycyclic compounds designed as ring-contracted or ring-expanded analogues of amantadine. inhibition of the wild-type (wt) m2 channel and the a/m2-s31n and a/m2-v27a mutant ion channels were measured in xenopus oocytes using two-electrode voltage clamp (tev) assays. several bisnoradam ... | 2011 | 21466220 |
| an intein-cassette integration approach used for the generation of a split tev protease activated by conditional protein splicing. | ligand-induced conditional protein splicing (cps) using a split intein allows the covalent reconstitution of a protein from two polypeptide fragments. the small molecule rapamycin binds to the fused fkbp and frb dimerizer domains and thereby induces folding of the split intein, which then removes itself in the trans-splicing reaction. cps has great potential for the experimental control of protein activity in living cells, however, only one such example was reported yet. this discrepancy is due ... | 2011 | 21487580 |
| hc-pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers. | abstract: background: rna silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. accordingly, plant viruses have evolved to produce counter defensive rna-silencing suppressors (rsss). these factors interfere in various ways with the rna silencing machinery in cells, and thereby disturb the microrna (mirna) mediated endogene regulation and induce developmental and morphological changes in plants. in this study we have explored these effects using ... | 2011 | 21507209 |
| helper component proteinase of genus potyvirus is an interaction partner of translation initiation factors eif(iso)4e and eif4e that contains a 4e binding motif. | the multifunctional helper component proteinase (hcpro) of potyviruses (genus potyvirus; potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eif4e and the isoform eif(iso)4e, which interact with viral genome-linked protein (vpg). here we show that hcpro of potato virus a (pva) interacts with both eif4e and eif(iso)4e, with interactions with eif(is ... | 2011 | 21525344 |
| the variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal. | recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. expression constructs with affinity tags often include an engineered protease site for tag removal. like other enzymes, the activities of proteases can be affected by buffer conditions. the buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. we examined the detergent sensitivity of six commonly-used proteases (enterokinase, f ... | 2011 | 21539919 |