Publications

domain isolation, expression, purification and proteolytic activity of the metalloprotease prtv from vibrio cholerae.	the metalloprotease prtv from vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. the protein belongs to the family of m6 proteases, with a characteristic zinc ion in the catalytic active site. prtv constitutes a 918 amino acids (102 kda) multidomain pre-pro-protein that so far has only been expressed in v. cholerae. structural studies require high amounts of soluble protein with high purity. previous attempts for recombinant expression have ...	2014	24492010
development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus.	tomato necrotic stunt virus (tonstv) is an emerging potyvirus that causes severe stunting to infected tomato plants. a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay was developed for sensitive detection of tonstv. the sensitivity of rt-lamp was comparable to that of conventional rt-pcr, with detection of tonstv in a reaction containing only 8 pg of total tomato rna or with 1:20,000 dilution of crude tissue extract. this assay was able to detect tonstv in a broad ra ...	2014	24503040
prokaryotic overexpression of tev-rhgh and characterization of its polyclonal antibody.	recombinant protein technology represents one of the best solutions to achieve rapid, efficient, and cost-effective protein expression and purification of therapeutic proteins. growth hormone (gh) is an excellent example of these proteins used in the therapy of hormone deficiencies. in this work, a plasmid, prset-tev-rhgh, has been constructed to overexpress recombinant human gh (rhgh) by cloning its gene downstream of an n-terminal 6 × his-tagged polypeptide (43 aa) in the t7 promoter-plasmid p ...	2014	24534464
shrinkage of genome size in a plant rna virus upon transfer of an essential viral gene into the host genome.	nonretroviral integrated rna viruses (nirvs) are genes of nonretroviral rna viruses found in the genomes of many eukaryotic organisms. nirvs are thought to sometimes confer virus resistance, meaning that they could impact spread of the virus in the host population. however, a nirv that is expressed may also impact the evolution of virus populations within host organisms. here, we experimentally addressed the evolution of a virus in a host expressing a nirv using tobacco etch virus (tev), a plant ...	2014	24558257
effects of the sequence characteristics of mirnas on multi-viral resistance mediated by single amirnas in transgenic tobacco.	artificial microrna (amirna) has become the preferred viral defence that can be induced in plants. in this study, nine amirna target sites were selected that were based on the sequence characteristics of natural mirnas in the cylindrical inclusion protein (ci), nuclear inclusion a protein (nia), nuclear inclusion b protein (nib), and coat protein (cp) genes of potato virus y (pvy(n)). these amirnas that exhibited high similarities to the sequences of pvy(n) and tev-sd1 were considered. to study ...	2014	24561715
expression of avian influenza virus (h5n1) hemagglutinin and matrix protein 1 in pichia pastoris and evaluation of their immunogenicity in mice.	the conventional avian influenza vaccines rely on development of neutralizing antibodies against the ha and na antigens. however, these antigens are highly variable, and hence there is a need for better vaccine candidates which would offer broader protection in animals. the m1 of avian influenza is another major structural protein that has conserved epitopes that are reported to induce cd8+ t cells and can contribute to protection against morbidity and mortality from influenza. hence in an effor ...	2014	24562978
site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation.	we described a rapid site-selective protein immobilization strategy on glass slides and magnetic nanoparticles, at either the n or c terminus, by a 2-cyanobenzothiazole (cbt)-cysteine (cys) condensation reaction. a terminal cysteine was generated at either terminus of a target protein by a combination of expressed protein ligation (epl) and tobacco etch virus protease (tevp) digestion, and was reacted with the cbt-solid support to immobilize the protein. according to microarray analysis, we foun ...	2014	24596243
specific requirement for translation initiation factor 4e or its isoform drives plant host susceptibility to tobacco etch virus.	in plants, eif4e translation initiation factors and their eifiso4e isoforms are essential susceptibility factors for many rna viruses, including potyviruses. mutations altering these factors are a major source of resistance to the viruses. the eif4e allelic series is associated with specific resistance spectra in crops such as capsicum annum. genetic evidence shows that potyviruses have a specific requirement for a given 4e isoform that depends on the host plant. for example, tobacco etch virus ...	2014	24645730
a new fusion protein platform for quantitatively measuring activity of multiple proteases.	recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (dal) as the fusion protein. it was based on the finding that a fused his6-tag could significantly decreases the activities of dal from e. coli (edal) and salmonella typhimurium (sdal). previously, we have shown that his6gst-tagged edal could be used t ...	2014	24649897
development of a multiplex bead-based assay to monitor dengue virus seroconversion.	dengue virus (denv) envelope protein is responsible for viral attachment to host cells and as such is a target of neutralizing antibody responses. however, the presence of envelope-specific antibodies against a given serotype may contribute to enhanced disease during secondary infection with another serotype. there is a need therefore for a standardized, high-throughput low-volume assay which permits the simultaneous screening of reactivity to multiple denv serotypes. here, we describe a method ...	2014	24696331
variability in mutational fitness effects prevents full lethal transitions in large quasispecies populations.	the distribution of mutational fitness effects (dmfe) is crucial to the evolutionary fate of quasispecies. in this article we analyze the effect of the dmfe on the dynamics of a large quasispecies by means of a phenotypic version of the classic eigen's model that incorporates beneficial, neutral, deleterious, and lethal mutations. by parameterizing the model with available experimental data on the dmfe of vesicular stomatitis virus (vsv) and tobacco etch virus (tev), we found that increasing mut ...	2014	24713667
linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with tev protease and autonomous n-terminal cyclization with glutaminyl cyclase in vivo.	overproduction of n-terminal pyroglutamate (pglu)-modified proteins utilizing escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the n-terminal glutaminyl or glutamyl residue, which is then converted into pglu catalyzed by the enzyme glutaminyl cyclase. herein we describe a new method for production of n-terminal pglu-containing proteins in vivo via intracellular self-clea ...	2014	24733552
resolution of telomere associations by trf1 cleavage in mouse embryonic stem cells.	telomere associations have been observed during key cellular processes such as mitosis, meiosis, and carcinogenesis and must be resolved before cell division to prevent genome instability. here we establish that telomeric repeat-binding factor 1 (trf1), a core component of the telomere protein complex, is a mediator of telomere associations in mammalian cells. using live-cell imaging, we show that expression of trf1 or yellow fluorescent protein (yfp)-trf1 fusion protein above endogenous levels ...	2014	24829382
trypanosoma brucei translation initiation factor homolog eif4e6 forms a tripartite cytosolic complex with eif4g5 and a capping enzyme homolog.	trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene-one-promoter arrangement. rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. posttranscriptional regulation of gene expression must thus play a larger role in these organisms. the eif4e homolog tbeif4e6 binds mrna cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. knockdow ...	2014	24839125
generation and characterization of nanobodies against rhgh expressed as sfgfp fusion protein.	growth hormone (gh) deficiencies are diagnosed in most children with short stature and treated with a long course of administrating expensive and daily doses of recombinant human gh (rhgh or somatropin®). this work describes for the first time the production of several gh specific nanobodies with great potential in the field of gh production and detection. nanobodies are the smallest intact antigen binders derived from heavy chain-only antibodies (hcabs) of camelids. they are very stable, highly ...	2014	24859761
experimental evolution of an emerging plant virus in host genotypes that differ in their susceptibility to infection.	this study evaluates the extent to which genetic differences among host individuals from the same species condition the evolution of a plant rna virus. we performed a threefold replicated evolution experiment in which tobacco etch potyvirus isolate at17b (tev-at17b), adapted to arabidopsis thaliana ecotype ler-0, was serially passaged in five genetically heterogeneous ecotypes of a. thaliana. after 15 passages we found that evolved viruses improved their fitness, showed higher infectivity and st ...	2014	24889935
his224 alters the r2 drug binding site and phe218 influences the catalytic efficiency of the metallo-β-lactamase vim-7.	metallo-β-lactamases (mbls) are the causative mechanism for resistance to β-lactams, including carbapenems, in many gram-negative pathogenic bacteria. one important family of mbls is the verona integron-encoded mbls (vim). in this study, the importance of residues asp120, phe218, and his224 in the most divergent vim variant, vim-7, was investigated to better understand the roles of these residues in vim enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking ...	2014	24913158
rescuing aggregation-prone proteins in escherichia coli with a dual his₆-mbp tag.	insolubility of recombinant proteins in escherichia coli is a major impediment to their production for structural and functional studies. one way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. e. coli maltose-binding protein (mbp) is widely recognized as a premier solubilizing agent. in this chapter, we describe how to construct dual his6-mbp-tagged fusion proteins by gateway(®) recombinational cloning and how to predict their yield and solubility. we al ...	2014	24943316
detection of protein-protein interactions using tandem affinity purification.	tandem affinity purification (tap) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. the approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. frequently two specific elution steps are also performed to increase the specificity of the overall technique. in the method detailed here, the two tags used are protein g and a short streptavidin binding peptide; however, many variations ...	2014	24943319
tobacco etch virus protein p1 traffics to the nucleolus and associates with the host 60s ribosomal subunits during infection.	the genus potyvirus comprises a large group of positive-strand rna plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. p1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. we infected plants with tobacco etch virus (tev; genus potyvirus) clones in which p1 was tagged with a fluorescent protein to track its expression and subcellular ...	2014	24991017
replication-associated proteins encoded by wheat dwarf virus act as rna silencing suppressors.	wheat dwarf virus (wdv) is an economically important and widespread single-stranded dna virus in the genus mastrevirus, family geminiviridae. so far, an rna silencing suppressor in the genus mastrevirus has not been reported. in this study, the viral replication-associated proteins of wdv, rep and repa proteins, were demonstrated to have rna silencing suppressor activities when expressed in agro-infiltrated leaves of nicotiana benthamiana line 16c, by their ability to inhibit posttranscriptional ...	2014	25016035
testing the independent action hypothesis of plant pathogen mode of action: a simple and powerful new approach.	the independent action hypothesis is a simple model of pathogen infection that can make many useful predictions on infection kinetics and, therefore, a number of different tests of independent action have been developed. however, some of these analyses are rather sophisticated, limiting their appeal to experimentalists, and it is also unclear how well the different tests perform. here, we developed and evaluated a simple and robust new test of independent action. our new test is based on using a ...	2015	25098495
soluble expression and purification of receptor activator of nuclear factor-kappa b ligand using escherichia coli.	receptor activator of nuclear factor-kappa b ligand (rankl) is a critical factor in osteoclastogenesis. it makes osteoclasts differentiate and multinucleate in bone remodeling. in the present study, rankl was expressed as a soluble maltose binding protein (mbp)-fusion protein using the escherichia coli maltose binding domain tag system (pmal) expression vector system. the host cell e. coli dh5α was cultured and induced by isopropyl β-d-1- thiogalactopyranoside for rrankl expression. cells were d ...	2015	25248982
an optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins.	head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. one way to obtain cyclic proteins via recombinant expression makes use of engineered intein tags, which are self-cleaving protein domains. in this approach, ph-induced self-cleavage of the n-terminal intein tag generates an n-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the c-terminal thioester formed by the second c-terminal intei ...	2014	25335928
molecular basis of calpain cleavage and inactivation of the sodium-calcium exchanger 1 in heart failure.	cardiac sodium (na(+))-calcium (ca(2+)) exchanger 1 (ncx1) is central to the maintenance of normal ca(2+) homeostasis and contraction. studies indicate that the ca(2+)-activated protease calpain cleaves ncx1. we hypothesized that calpain is an important regulator of ncx1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of ncx1 in the heart. ncx1 full-length protein and a 75-kda ncx1 fragment along with calpain ...	2014	25336645
the pcri system: a vector collection for recombinant protein expression and purification.	a major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. here we introduce a vector collection, the pcri system, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (escherichia coli, bacillus subtilis, and pichia pastoris). by using a single pol ...	2014	25386923
differential disease symptoms and full-length genome sequence analysis for three strains of tobacco etch virus.	tobacco etch virus (tev) strains hat, mex21, and n have been the focus of numerous studies to dissect a host resistance mechanism in capsicum spp. little is known, however, about their general pathogenicity and genomic sequence data are not available on the tev strains mex21 and n. four nicotiana spp. were evaluated after inoculation with each tev strain. nicotiana tabacum 'kentucky 14' and n. clevelandii plants expressed varied systemic symptoms dependent on the tev strain; however, disease sev ...	2015	25425495
degradation of c-terminal tag sequences on domain antibodies purified from e. coli supernatant.	expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. however, as the fusion peptides most often are flexible appendages at the n- or c-terminal, proteolytic cleavage may result in removal of the tag sequence. here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. the tag sequenc ...	2014	25426869
the yhhn protein of legionella pneumophila is a lysoplasmalogenase.	lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. we recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as tmem86b, an integral membrane protein that is a member of the yhhn family found in numerous species of eukaryotes and bacteria. to test the hypothesis that bacterial yhhn proteins also function as lysoplasmalogenase enzymes, we c ...	2014	25445671
genome sequence of a virus isolate from tamarillo (solanum betaceum) in colombia: evidence for a new potyvirus.	based on the results of a deep sequencing transcriptome study of tamarillo (solanum betaceum), we report the genome sequence of a virus from this host plant. since this probably represents a new member of the genus potyvirus, the name tamarillo leaf malformation virus (talmv) has been proposed. phylogenetic analysis reveals that talmv is the closest relative of colombian datura virus (cdv), followed by three other potyviruses: tobacco etch virus, potato virus a and tobacco vein mottling virus. t ...	2015	25466572
a tandem affinity purification tag of tga2 for isolation of interacting proteins in arabidopsis thaliana.	tandem affinity purification (tap) tagging provides a powerful tool for isolating interacting proteins in vivo. tap-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. type ii bzip transcription factors (tga2, tga5 and tga6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. although proteins interacting with these transcription factors have been identified through genetic and ye ...	2014	25482810
amyloid-like assembly of the low complexity domain of yeast nab3.	termination of transcription of short non-coding rnas is carried out in yeast by the nab3-nrd1-sen1 complex. nab3 and nrd1 are hnrnp-like proteins that dimerize and bind rna with sequence specificity. we show here that an essential region of nab3 that is predicted to be prion-like based upon its sequence bias, formed amyloid-like filaments. a similar region from nrd1 also assembled into filaments in vitro. the purified nab3 domain formed a macroscopic gel whose lattice organization was observed ...	2015	25611193
temporal dynamics of intrahost molecular evolution for a plant rna virus.	populations of plant rna viruses are highly polymorphic in infected plants, which may allow rapid within-host evolution. to understand tobacco etch potyvirus (tev) evolution, longitudinal samples from experimentally evolved populations in the natural host tobacco and from the alternative host pepper were phenotypically characterized and genetically analyzed. temporal and compartmental variabilities of tev populations were quantified using high throughput illumina sequencing and population geneti ...	2015	25660377
the sh3 regulatory domain of the hematopoietic cell kinase hck binds elmo via its polyproline motif.	eukaryotic engulfment and cell motility (elmo) proteins form an evolutionary conserved family of regulators involved in small gtpase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the downstream of crk (dock) family members. gathered data strongly suggest that dock activation by elmo and the subsequent signaling result from a subtle balance in the binding of partners to elmo. among its putative upward modulators, the hematopoietic cell kinase ...	2015	25737835
rna-dependent rna polymerase (nib) of the potyviruses is an avirulence factor for the broad-spectrum resistance gene pvr4 in capsicum annuum cv. cm334.	potyviruses are one of the most destructive viral pathogens of solanaceae plants. in capsicum annuum landrace cm334, a broad-spectrum gene, pvr4 is known to be involved in resistance against multiple potyviruses, including pepper mottle virus (pepmov), pepper severe mosaic virus (pepsmv), and potato virus y (pvy). however, a potyvirus avirulence factor against pvr4 has not been identified. to identify the avirulence factor corresponding to pvr4 in potyviruses, we performed agrobacterium-mediated ...	2015	25760376
single cell fret analysis for the identification of optimal fret-pairs in bacillus subtilis using a prototype mem-flim system.	protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. in contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. one approach to study protein-protein interactions in vivo is via förster resonance energy transfer (fret). here, fret efficiency of selected fret-pairs was studied at the single cell ...	2015	25886351
yeast endoplasmic reticulum sequestration screening for the engineering of proteases from libraries expressed in yeast.	there is significant interest in engineering proteases with desired proteolytic properties. we describe a high-throughput fluorescence-activated cell sorting (facs) assay for detecting altered proteolytic activity of protease in yeast, at the single cell level. this assay relies on coupling yeast endoplasmic reticulum (er) retention, yeast surface display, and facs analysis. the method described here allows facile screening of large libraries, and of either protease or substrate variants, includ ...	2015	26060071
handicap-recover evolution leads to a chemically versatile, nucleophile-permissive protease.	mutation of the tobacco etch virus (tev) protease nucleophile from cysteine to serine causes an approximately ∼10(4) -fold loss in activity. ten rounds of directed evolution of the mutant, tev(ser) , overcame the detrimental effects of nucleophile exchange to recover near-wild-type activity in the mutant tev(ser) x. rather than respecialising tev to the new nucleophile, all the enzymes along the evolutionary trajectory also retained the ability to use the original cysteine nucleophile. therefore ...	2015	26097079
engineered cellular gene-replacement platform for selective and inducible proteolytic profiling.	cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated dnase (cad). cad is activated when caspases cleave its endogenous inhibitor icad, resulting in the characteristic dna laddering of apoptosis. we describe a posttranscriptional gene replac ...	2015	26106156
halotag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors.	the production of recombinant proteins such as the fibroblast growth factors (fgfs) is the key to establishing their function in cell communication. the production of recombinant fgfs in e. coli is limited, however, due to expression and solubility problems. halotag has been used as a fusion protein to introduce a genetically-encoded means for chemical conjugation of probes. we have expressed 11 fgf proteins with an n-terminal halotag, followed by a tobacco etch virus (tev) protease cleavage sit ...	2015	26137434
a potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein.	plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. among them, a system derived from tobacco etch virus (tev; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. this work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. we constructed tev clones in which green fluorescent protein (gfp), with a chloroplast transit pep ...	2015	26147811
evaluating the within-host fitness effects of mutations fixed during virus adaptation to different ecotypes of a new host.	the existence of genetic variation for resistance in host populations is assumed to be essential to the spread of an emerging virus. models predict that the rate of spread slows down with the increasing frequency and higher diversity of resistance alleles in the host population. we have been using the experimental pathosystem arabidopsis thaliana-tobacco etch potyvirus (tev) to explore the interplay between genetic variation in host's susceptibility and virus diversity. we have recently shown th ...	2015	26150658
versatile vector suite for the extracytoplasmic production and purification of heterologous his-tagged proteins in lactococcus lactis.	recent studies have shown that the gram-positive bacterium lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of n- or c-terminally hexa-histidine (his6)-tagged ...	2015	26160391
phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.	bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. we propose a proof-of-principal phage-based scheme to enable mu ...	2015	26245682
biophysical characterization of a vaccine candidate against hiv-1: the transmembrane and membrane proximal domains of hiv-1 gp41 as a maltose binding protein fusion.	the membrane proximal region (mpr, residues 649-683) and transmembrane domain (tmd, residues 684-705) of the gp41 subunit of hiv-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. several structures of the trimeric membrane proximal external region (residues 662-683) of mpr have been reported at the atomic level; however, the atomic structure of the tmd still remains unknown. to elucidate the structur ...	2015	26295457
engineered tobacco etch virus (tev) protease active in the secretory pathway of mammalian cells.	tobacco etch virus protease (tevp) is a unique endopeptidase with stringent substrate specificity. tevp has been widely used as a purified protein for in vitro applications, but also as a biological tool directly expressing it in living cells. to adapt the protease to diverse applications, several tevp mutants with different stability and enzymatic properties have been reported. herein we describe the development of a novel engineered tevp mutant designed to be active in the secretory pathway. w ...	2015	26327323
the impact of high-order epistasis in the within-host fitness of a positive-sense plant rna virus.	rna viruses are the main source of emerging infectious diseases because of the evolutionary potential bestowed by their fast replication, large population sizes and high mutation and recombination rates. however, an equally important property, which is usually neglected, is the topography of the fitness landscape. how many fitness maxima exist and how well they are connected is especially interesting, as this determines the number of accessible evolutionary pathways. to address this question, we ...	2015	26344415
rapid large-scale purification of myofilament proteins using a cleavable his6-tag.	with the advent of high-throughput dna sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. as the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. however, producing large quantities of myofilament proteins can be time consuming a ...	2015	26386113
fusion of genomic, proteomic and phenotypic data: the case of potyviruses.	data fusion has been widely applied to analyse different sources of information, combining all of them in a single multivariate model. this methodology is mandatory when different omic data sets must be integrated to fully understand an organism using a systems biology approach. here, a data fusion procedure is presented to combine genomic, proteomic and phenotypic data sets gathered for tobacco etch virus (tev). the genomic data correspond to random mutations inserted in most viral genes. the p ...	2016	26593691
single-step affinity and cost-effective purification of recombinant proteins using the sepharose-binding lectin-tag from the mushroom laetiporus sulphureus as fusion partner.	previous research showed that a lectin from the mushroom laetiporus sulphureus, designed lsl, bound to sepharose and could be eluted by lactose. in this study, by taking advantage of the strong affinity of lsl-tag for sepharose, we developed a single-step purification method for lsl-tagged fusion proteins. we utilized unmodified sepharose-4b as a specific adsorbent and 0.2 m lactose solution as an elution buffer. fusion proteins of lsl-tag and porcine circovirus capsid protein, designated lsl-ca ...	2016	26616099
distribution of mutational fitness effects and of epistasis in the 5' untranslated region of a plant rna virus.	understanding the causes and consequences of phenotypic variability is a central topic of evolutionary biology. mutations within non-coding cis-regulatory regions are thought to be of major effect since they affect the expression of downstream genes. to address the evolutionary potential of mutations affecting such regions in rna viruses, we explored the fitness properties of mutations affecting the 5'-untranslated region (utr) of a prototypical member of the picorna-like superfamily, tobacco et ...	2015	26643527
simultaneous selection and counter-selection for the directed evolution of proteases in e. coli using a cytoplasmic anchoring strategy.	with the goal of generating new enzymes that can cleave custom sequences, this article describes a selection strategy for evolving proteases with desirable characteristics. positive selection and counter-selection are combined to select for and against specified cleavage sequences simultaneously. cleavage of the positive selection sequence permits e. coli growth, and cleavage of the counter-selection sequence slows growth. growth occurs when cleavage of the positive selection sequence releases β ...	2016	26666461
preparation of specific polyclonal antibody against the recombinant mutacin produced by sfgfp fusion protein technology.	mutacin i, a bacteriocin produced by streptococcus mutans, displays an antimicrobial activity against many gram positive and some gram negative bacteria. because of its medical importance, production of this short peptide in large scale for future applications is a significant challenge. this work described the improvement of a novel system to produce the recombinant mutacin using fusion protein technology. the short peptide was expressed directly as a fusion protein with a superfolder form of t ...	2015	26668664
interaction network of tobacco etch potyvirus nia protein with the host proteome during infection.	the genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements. however, the interaction networks between viruses and host factors are poorly understood. the genus potyvirus is the largest group of plus-strand rna viruses infecting plants. potyviral nuclear inclusion a (nia) plays many roles during infection. nia is a polyprotein consisting of two domains, viral protein genome-linked (vpg) an ...	2016	26830344
a tilling approach to generate broad-spectrum resistance to potyviruses in tomato is hampered by eif4e gene redundancy.	genetic resistance to pathogens is important for sustainable maintenance of crop yields. recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. tomato (solanum lycopersicum) and related species offer a model in which natural and tilling-induced potyvirus resistance alleles may be compared. for resistance based on translation initiation factor eif4e1, we confirm that the natural allele sh-eif4e1(pi24)-pot1, isolated fro ...	2016	26850324
a transcription activator-like effector (tale) induction system mediated by proteolysis.	simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. transcription activator-like effectors (tales) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable dna-binding specificity. in this work we expanded the versatility of these transcription factors to cre ...	2016	26854666
cleavage of fusion proteins on the affinity resins using the tev protease variant.	it is documented that the tobacco etch virus protease (tevp) variant tevp(3m) is less efficient in cleaving the fusion protein bound to ni-nta resin at relatively low temperature. here, we determined that, using the gfp fusion substrate bound to ni-nta or strep-tactin agarose, activity of the tevp(5m) variant was higher than that of the other tevp construct, and about 15% higher than that of the tevp(3m). the gst fusion proteins immobilized on strep-tactin agarose or glutathione sepharose were e ...	2016	26876021
tandem affinity purification in drosophila heads and ovaries.	tandem affinity purification (tap) (pugi et al., 2001; rigaut et al., 1999) is a method that uses a tagging approach of a target protein of interest for a two-step purification scheme in order to pull down protein complexes under native conditions and expression levels. the tap tag consists of three components: a calmodulin-binding peptide, a tobacco etch virus (tev) protease cleavage site and protein a which is an immunoglobulin g (igg)-binding domain. this protocol was modified from the origin ...	2012	27042686
activity of the human rhinovirus 3c protease studied in various buffers, additives and detergents solutions for recombinant protein production.	proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. in recent years, great interest has been seen in use of the human rhinovirus 3c protease owing to its stringent sequence specificity and enhanced activity. like other proteases, activity of the human rhinovirus 3c protease can be affected in part by the buffer components and additives that are generally emp ...	2016	27093053
a dual protease approach for expression and affinity purification of recombinant proteins.	we describe a new method for affinity purification of recombinant proteins using a dual protease protocol. escherichia coli maltose binding protein (mbp) is employed as an n-terminal tag to increase the yield and solubility of its fusion partners. the mbp moiety is then removed by rhinovirus 3c protease, prior to purification, to yield an n-terminally his6-tagged protein. proteins that are only temporarily rendered soluble by fusing them to mbp are readily identified at this stage because they w ...	2016	27105777
the transcriptomics of an experimentally evolved plant-virus interaction.	models of plant-virus interaction assume that the ability of a virus to infect a host genotype depends on the matching between virulence and resistance genes. recently, we evolved tobacco etch potyvirus (tev) lineages on different ecotypes of arabidopsis thaliana, and found that some ecotypes selected for specialist viruses whereas others selected for generalists. here we sought to evaluate the transcriptomic basis of such relationships. we have characterized the transcriptomic responses of five ...	2016	27113435
new high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in escherichia coli and salmonella enterica.	galloway et al. recently described a method to alter vectors to include type iis restriction enzymes for high efficiency cloning. utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the type iis restriction enzyme, bspqi. use of this enzyme increased the rate of cloning success to >97% efficiency. l(+)-arabinose-inducible complementation vectors and overexpression vectors ...	2016	27234933
tobacco etch virus protease: a shortcut across biotechnologies.	about thirty years ago, studies on the rna genome of tobacco etch virus revealed the presence of an efficient and specific protease, called tobacco etch virus protease (tevp), that was part of the nuclear inclusion a (nia) enzyme. tevp is an efficient and specific protease of 27kda that has become a valuable biotechnological tool. nowadays tevp is a unique endopeptidase largely exploited in biotechnology from industrial applications to in vitro and in vivo cellular studies. a number of tevp muta ...	2016	27312702
generation of artificial n-end rule substrate proteins in vivo and in vitro.	in order to determine the stability of a protein or protein fragment dependent on its n-terminal amino acid, and therefore relate its half-life to the n-end rule pathway of targeted protein degradation (nerd), non-methionine (met) amino acids need to be exposed at their amino terminal in most cases. per definition, at this position, destabilizing residues are generally unlikely to occur without further posttranslational modification of immature (pre-)proproteins. moreover, almost exclusively, st ...	2016	27424746
data for the co-expression and purification of human recombinant camkk2 in complex with calmodulin in escherichia coli.	calcium/calmodulin-dependent kinase kinase 2 (camkk2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. here, we describe the expression in escherichia coli and the purification protocol for the following constructs: full-length camkk2 in complex with cam, camkk2 'apo', camkk2 (165-501) in complex with cam, and the camkk2 f267g mutant. the protocols described have been optimized for maximum yield and purity with minimal purification steps required and ...	2016	27508226
efficient escape from local optima in a highly rugged fitness landscape by evolving rna virus populations.	predicting viral evolution has proven to be a particularly difficult task, mainly owing to our incomplete knowledge of some of the fundamental principles that drive it. recently, valuable information has been provided about mutation and recombination rates, the role of genetic drift and the distribution of mutational, epistatic and pleiotropic fitness effects. however, information about the topography of virus' adaptive landscapes is still scarce, and to our knowledge no data has been reported s ...	2016	27534955
a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease.	recombinant protein purification remains to be a major challenge in biotechnology and medicine. in this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (tevp). after construction of an n-terminal elk16 peptide fusion expression vector, we expressed elk16-tevp fusion protein in e. coli. sds-page analysis showed that elk16-tevp was expressed as active protein aggregates which could be purified to 91% purity with ...	2016	27546453
effect of host species on the topography of fitness landscape for a plant rna virus.	adaptive fitness landscapes are a fundamental concept in evolutionary biology that relate the genotype of individuals with their fitness. at the end, the evolutionary fate of evolving populations depends on the topography of the landscape, that is, the number of accessible mutational pathways and of possible fitness peaks (i.e, adaptive solutions). for long time, fitness landscapes were only theoretical constructions due to a lack of precise information on the mapping between genotypes and pheno ...	2016	27581976
predicting the stability of homologous gene duplications in a plant rna virus.	one of the striking features of many eukaryotes is the apparent amount of redundancy in coding and non-coding elements of their genomes. despite the possible evolutionary advantages, there are fewer examples of redundant sequences in viral genomes, particularly those with rna genomes. the factors constraining the maintenance of redundant sequences in present-day rna virus genomes are not well known. here, we use tobacco etch virus, a plant rna virus, to investigate the stability of genetically r ...	2016	27604880
a non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the hiv-1 pr55(gag) polyprotein alters nucleic acid binding properties.	hiv gag (pr55(gag)), a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (hiv), is an active target of antiretroviral inhibitor development. however, highly pure, stable, recombinant pr55(gag) has been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. stability has been improved by using a construct that omits the p6 domain (δp6). in vivo, p ...	2017	27721079
genetic variation in fitness within a clonal population of a plant rna virus.	a long-standing observation in evolutionary virology is that rna virus populations are highly polymorphic, composed by a mixture of genotypes whose abundances in the population depend on complex interaction between fitness differences, mutational coupling and genetic drift. it was shown long ago, though in cell cultures, that most of these genotypes had lower fitness than the population they belong, an observation that explained why single-virion passages turned on muller's ratchet while very la ...	2016	27774299
protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting.	proteases are involved in fundamental biological processes and are important tools in both biotechnological and biomedical research. an important property of proteases is to discriminate among potential substrates. here, a new method for substrate profiling of proteases is presented. the substrates are displayed between two anti-idiotypic affinity domains on the gram-positive bacterium staphylococcus carnosus. the first domain functions as a reporter tag and has affinity for a labeled reporter p ...	2017	27783465
synthesis of fe3o4@nickel-silicate core-shell nanoparticles for his-tagged enzyme immobilizing agents.	immobilizing enzymes on artificially fabricated carriers for their efficient use and easy removal from reactants has attracted enormous interest for decades. specifically, binding platforms using inorganic nanoparticles have been widely explored because of the benefits of their large surface area, easy surface modification, and high stability in various ph and temperatures. herein, we fabricated fe3o4 encapsulated 'sea-urchin' shaped nickel-silicate nanoparticles with a facile synthetic route. t ...	2016	27831938
simple and efficient production and purification of mouse myelin oligodendrocyte glycoprotein for experimental autoimmune encephalomyelitis studies.	multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns), thought to occur as a result of autoimmune responses targeting myelin. experimental autoimmune encephalomyelitis (eae) is the most common animal model of cns autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant cd4(+) t cell epitopes of myelin proteins. however, b cells recognize unprocessed protein directly, and immunization with short peptide d ...	2016	27842340
self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce igg2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.	assembly of recombinant capsid proteins into virus-like particles (vlps) still represents an interesting challenge in virus-based nanotechnologies. the structure of vlps has gained importance for the development and design of new adjuvants and antigen carriers. the potential of tobacco etch virus capsid protein (tev cp) as adjuvant has not been evaluated to date.	2016	27894314
assessment of the fusion tags on increasing soluble production of the active tev protease variant and other target proteins in e. coli.	in this study, five fusion tags affecting soluble production and cleavage activity of the tobacco etch virus (tev) protease (tevp) variant in escherichia coli strains bl21 (de3) and rosetta™ (de3) are investigated. combination of the augmenting rare transfer rnas (trnas) and the fused expressivity tag (n-terminal seven amino acid residues of e. coli translation initiation factor ii) promotes the soluble tevp partner expressed at relatively high level. attachment of the maltose-binding protein (m ...	2016	27988855
high virulence does not necessarily impede viral adaptation to a new host: a case study using a plant rna virus.	theory suggests that high virulence could hinder between-host transmission of microparasites, and that virulence therefore will evolve to lower levels. alternatively, highly virulent microparasites could also curtail host development, thereby limiting both the host resources available to them and their own within-host effective population size. in this case, high virulence might restrain the mutation supply rate and increase the strength with which genetic drift acts on microparasite populations ...	2017	28103791
unusual fusion proteins of hiv-1.	despite its small genome size, the human immunodeficiency virus 1 (hiv-1) is one of the most successful pathogens and has infected more than 70 million people worldwide within the last decades. in total, hiv-1 expresses 16 canonical proteins from only nine genes within its 10 kb genome. expression of the structural genes gag, pol, and env, the regulatory genes rev and tat and the accessory genes vpu, nef, vpr, and vif enables assembly of the viral particle, regulates viral gene transcription, an ...	2016	28119676
2b or not 2b: experimental evolution of functional exogenous sequences in a plant rna virus.	horizontal gene transfer (hgt) is pervasive in viruses and thought to be a key mechanism in their evolution. on the other hand, strong selective constraints against increasing genome size are an impediment for hgt, rapidly purging horizontally transferred sequences and thereby potentially hindering evolutionary innovation. here, we explore experimentally the evolutionary fate of viruses with simulated hgt events, using the plant rna virus tobacco etch virus (tev), by separately introducing two f ...	2017	28137747
rewiring carotenoid biosynthesis in plants using a viral vector.	plants can be engineered to sustainably produce compounds of nutritional, industrial or pharmaceutical relevance. this is, however, a challenging task as extensive regulation of biosynthetic pathways often hampers major metabolic changes. here we describe the use of a viral vector derived from tobacco etch virus to express a whole heterologous metabolic pathway that produces the health-promoting carotenoid lycopene in tobacco tissues. the pathway consisted in three enzymes from the soil bacteria ...	2017	28139696
hirudin as a novel fusion tag for efficient production of lunasin in escherichia coli.	fusion expression provides an effective means for the biosynthesis of longer peptides in escherichia coli. however, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of the commercial affinity resins. herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tev protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acids model peptide lunasin in e. coli at pre ...	2017	28151045
improved tandem affinity purification tag and methods for isolation of proteins and protein complexes from schizosaccharomyces pombe.	the tandem affinity purification (tap) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. proteins purified via the tap tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. the original tap tag contains a calmodulin-binding peptide and the igg-binding domain from protein a separated by a tobacco etch virus (tev) protease cleavage site. ...	2017	28250214
identification of host factors potentially involved in rtm-mediated resistance during potyvirus long distance movement.	the long distance movement of potyviruses is a poorly understood step of the viral cycle. only factors inhibiting this process, referred to as "restricted tev movement" (rtm), have been identified in arabidopsis thaliana. on the virus side, the potyvirus coat protein (cp) displays determinants required for long-distance movement and for rtm-based resistance breaking. however, the potyvirus cp was previously shown not to interact with the rtm proteins. we undertook the identification of arabidops ...	2017	28251380
temporally precise labeling and control of neuromodulatory circuits in the mammalian brain.	few tools exist to visualize and manipulate neurons that are targets of neuromodulators. we present itango, a light- and ligand-gated gene expression system based on a light-inducible split tobacco etch virus protease. cells expressing the itango system exhibit increased expression of a marker gene in the presence of dopamine and blue-light exposure, both in vitro and in vivo. we demonstrated the itango system in a behaviorally relevant context, by inducing expression of optogenetic tools in neu ...	2017	28369042
chimeric flock house virus protein a with endoplasmic reticulum-targeting domain enhances viral replication and virus-like particle trans-encapsidation in plants.	flock house virus (fhv) rna can be trans-encapsidated, entirely in planta, by tobacco mosaic virus coat protein to form virus-like particles (vlps). vaccination with these vlps leads to strong antigen expression in mice and immune-activation. we hypothesize that creating an additional cellular site for replication and/or trans-encapsidation might significantly improve the final output of trans-encapsidated product. fhv protein a was engineered to target the endoplasmic reticulum (er) via a heter ...	2017	28437636
tobacco etch virus protease mediating cleavage of the cellulose-binding module tagged colored proteins immobilized on the regenerated amorphous cellulose.	in this study, four fusion proteins were designed, in which the n-terminal cellulose-binding module as the affinity tag was immobilized on the regenerated amorphous cellulose (rac), and the release of the c-terminal colored proteins was detected easily and rapidly after on-resin cleavage using the free tobacco etch virus protease (tevp) variant, or the immobilized cognate protease with a binding capacity of up to 220 mg protein per gram of rac. the enhanced stability and repetitive use of the im ...	2017	28439671
removal of affinity tags with tev protease.	although affinity tags are highly effective tools for the expression and purification of recombinant proteins, they generally need to be removed prior to structural and functional studies. this chapter describes a simple method for overproducing a soluble form of a stable variant of tobacco etch virus (tev) protease in escherichia coli and a protocol for purifying it to homogeneity so that it can be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoprote ...	2017	28470608
fusion expression of the pgla-am1 with native structure and evaluation of its anti-helicobacter pylori activity.	helicobacter pylori (h. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. the antimicrobial peptide pgla-am1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. in this study, we investigated whether it has anti-h. pylori activity for the further development of anti-h. pylori drugs to replace existing antibiotics. however, the natural antimicrobial peptide pgla-am1 shows a low yield an ...	2017	28488117
modularly constructed synthetic granzyme b molecule enables interrogation of intracellular proteases for targeted cytotoxicity.	targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. however, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. in contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. here, we report a protein-based thera ...	2017	28510446
expression and purification of recombinant proteins in escherichia coli with a his6 or dual his6-mbp tag.	rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in escherichia coli. affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. yet insolubility of recombinant proteins in e. coli remains a persistent problem. one way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. e. coli maltose-binding pr ...	2017	28573567
genetic encoding of photocaged tyrosines with improved light-activation properties for the optical control of protease function.	we genetically encoded three new caged tyrosine analogues with improved photochemical properties by using an engineered pyrrolysyl-trna synthetase/trnacua pair in bacterial and mammalian cells. we applied the new tyrosine analogues to the photoregulation of firefly luciferase by caging its key tyrosine residue, tyr340, and observed excellent off-to-on light switching. this reporter was then used to evaluate the activation rates of the different light-removable protecting groups in live cells. we ...	2017	28608946
expression and purification of swine rag2 in e. coli for production of porcine rag2 polyclonal antibodies.	recombination activating gene 2 (rag2) is necessary for immature b cell differentiation. antibodies to human and rabbit rag2 are currently commercially available, but antibodies to swine rag remain unavailable to date. in this study, the swine rag2 genes sequence was synthesized and then cloned into a pet-28a vector. the recombinant fusion protein was successfully expressed in e. coli, purified through nickel column chromatography, and further digested with tobacco etch virus protease. the cleav ...	2017	28644752
a novel expression vector for the secretion of abaecin in bacillus subtilis.	this study aimed to describe a bacillus subtilis expression system based on genetically modified b. subtilis. abaecin, an antimicrobial peptide obtained from apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. for the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter pglv, and a mature beta-glucanase signal peptide. also, a b. subtilis expression system was constructe ...	2017	28651889
protein-protein interaction: tandem affinity purification in bacteria.	the discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. here we describe the protocol to perform tandem affinity purification (tap) in bacteria, which enables the identification of the partners of a bait protein under native conditions. this ...	2017	28667616
binding of hydroxycitrate to human atp-citrate lyase.	hydroxycitrate from the fruit of garcinia cambogia [i.e. (2s,3s)-2-hydroxycitrate] is the best-known inhibitor of atp-citrate lyase. well diffracting crystals showing how the inhibitor binds to human atp-citrate lyase were grown by modifying the protein. the protein was modified by introducing cleavage sites for tobacco etch virus protease on either side of a disordered linker. the protein crystallized consisted of residues 2-425-enlyfq and s-488-810 of human atp-citrate lyase. (2s,3s)-2-hydroxy ...	2017	28777081
recombinant scorpion toxins: focus on four-disulfide peptide blockers of kv1-channels.	we have recently developed a simple and effective bioengineering approach to large-scale production of alpha-ktx, peptide toxins from scorpion venoms, that block voltage-gated potassium channels with high affinity and specificity. this approach was successfully approved for different peptides containing three disulfide bonds. to extend this method to production of peptide toxins with four disulfide bridges, in particular, maurotoxin and hetlaxin, appropriate conditions of a cleavage reaction wit ...	2017	28857644