Publications

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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 ...201121267463
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 ...201121404008
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 ...201121295005
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 ...201121359142
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 ...201121414287
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 ...201121454607
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 ...201121466220
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 ...201121487580
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 ...201020631085
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 ...201020851955
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 ...201020308068
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 ...201020201562
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 ...201020862670
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 ...201021055470
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 ...201020658971
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 ...201020478894
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 ...201020667575
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 ...201020721950
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 ...201019945728
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 ...201020010833
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 ...201020064616
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 ...201020439778
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 ...201019603287
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 ...201020152868
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 ...201020408810
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.201021190544
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 ...201019906792
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 ...201020091191
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 ...201020148412
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 ...201020182554
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 ...201020208176
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 ...201020304006
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 ...201020376682
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 ...201021126542
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.201020378974
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 ...201020723762
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 ...201020723624
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 ...200919521826
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, ...200918983922
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 ...200918988025
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 ...200918683262
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 ...200919358526
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 ...200918937642
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 ...200919318686
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 ...200919322647
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 ...200919324646
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 ...200919244328
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 ...200919401679
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), ...200919453166
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 ...200919469531
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 ...200918988033
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 ...200919013248
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 ...200919152045
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 ...200919167339
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 ...200919198843
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 ...200919544035
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 ...200918845260
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 ...200919737103
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 ...200919530081
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 ...200919827097
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.200919830250
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 ...200919858189
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 ...200819226768
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 ...200818765302
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 ...200818982293
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 ...200818826949
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 ...200818855760
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 ...200818682245
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 ...200818684336
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 ...200818692164
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 ...200818780745
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 ...200818375143
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 ...200818226920
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 ...200818047580
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 ...200818063580
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 ...200818182024
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 ...200818280752
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 ...200818295882
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 ...200818457437
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 ...200818476830
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 ...200818539609
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 ...200818548328
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 ...200818564369
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 ...200818045881
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 ...200818595734
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 ...200818024078
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 ...200818599143
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 ...200818370252
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 ...200717566292
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- ...200717352916
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. ...200717406599
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 ...200717489638
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 ...200717662848
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 ...200717898073
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 ...200716919473
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 ...200717344305
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 ...200717616520
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 ...200717626049
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 ...200716964623
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 ...200717666392
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