Publications

analysis of the autoproteolytic activity of the recombinant helper component proteinase from zucchini yellow mosaic virus.	the multifunctional helper component proteinase (hc-pro) of potyviruses contains an autoproteolytic function that, together with the protein 1 (p1) and nia proteinase, processes the polyprotein into mature proteins. in this study, we analysed the autoproteolytic active domain of zucchini yellow mosaic virus (zymv) hc-pro. several escherichia coli-expressed mbp:hc-pro:gfp mutants containing deletions or point mutations at either the n- or c-terminus of the hc-pro protein were examined. our result ...	2011	21871010
a cell-based protein-protein interaction method using a permuted luciferase reporter.	we have developed a novel cell-based protein-protein interaction assay method. the method relies on conversion of an inactive permuted luciferase containing a tobacco etch virus protease (tev) cleavage sequence fused onto protein (a) to an active luciferase upon interaction and cleavage by another protein (b) fused with the tev protease. we demonstrate assay applicability for ligand-induced protein-protein interactions including g-protein coupled receptors, receptor tyrosine kinases and nuclear ...	2011	22207892
Effect of host species on the distribution of mutational fitness effects for an RNA virus.	Knowledge about the distribution of mutational fitness effects (DMFE) is essential for many evolutionary models. In recent years, the properties of the DMFE have been carefully described for some microorganisms. In most cases, however, this information has been obtained only for a single environment, and very few studies have explored the effect that environmental variation may have on the DMFE. Environmental effects are particularly relevant for the evolution of multi-host parasites and thus fo ...	2011	22125497
multihost experimental evolution of a plant rna virus reveals local adaptation and host-specific mutations.	for multihost pathogens, adaptation to multiple hosts has important implications for both applied and basic research. at the applied level, it is one of the main factors determining the probability and the severity of emerging disease outbreaks. at the basic level, it is thought to be a key mechanism for the maintenance of genetic diversity both in host and pathogen species. using tobacco etch potyvirus (tev) and four natural hosts, we have designed an evolution experiment whose strength and nov ...	2011	22319146
a new tagged-tev protease: construction, optimisation of production, purification and test activity.	the tobacco etch virus (tev) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity. in this work, we present a new recombinant form of tev termed streptag ii-tev for high-level production and purification of tev protease from escherichia coli and compare it to the hexahistidine (6xhis) tagged version of tev. the effects of varying the host strain, the bacterial induction temperature (25, 30 and 37°c) and the iptg inducer concent ...	2011	20817099
effect of lead (pb) on the systemic movement of rna viruses in tobacco (nicotiana tabacum var. turkish).	effect of various lead (pb) concentrations on the systemic movement of rna viruses was examined in tobacco plants. prior to inoculation, plants were grown hydroponically for 6 days in hoagland's solution supplemented with five concentrations of lead nitrate [pb(no(3))(2)]: 0.0 (control), 10, 15, 50, and 100 µm. four different rna viruses with different cell-to-cell movement mechanisms were used. two weeks after inoculation lower and upper leaves of each treatment were harvested and examined for ...	2011	21404008
predictive mutagenesis of ligation-independent cloning (lic) vectors for protein expression and site-specific chemical conjugation.	ligation-independent cloning (lic) allows for cloning of dna constructs independent of insert restriction sites and ligases. however, any required mutations are typically introduced by additional, time-consuming steps. we present a rapid, inexpensive method for mutagenesis in the 5' lic site of expression constructs and report on the construction of expression vectors with n-terminal serine, cysteine, threonine, or tyrosine residues after tobacco etch virus (tev) protease cleavage. in a practica ...	2011	21414287
differences in accumulation and virulence determine the outcome of competition during tobacco etch virus coinfection.	understanding the evolution of virulence for rna viruses is essential for developing appropriate control strategies. although it has been usually assumed that virulence is a consequence of within-host replication of the parasite, viral strains may be highly virulent without experiencing large accumulation as a consequence of immunopathological host responses. using two strains of tobacco etch potyvirus (tev) that show a negative relationship between virulence and accumulation rate, we first expl ...	2011	21423618
a "two-hit" hypothesis for inclusion formation by carboxyl-terminal fragments of tdp-43 protein linked to rna depletion and impaired microtubule-dependent transport.	carboxyl-terminal fragments (ctfs) of tdp-43 aggregate to form the diagnostic signature inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the biological significance of these ctfs and how they are generated remain enigmatic. to address these issues, we engineered mammalian cells with an inducible tobacco etch virus (tev) protease that cleaves tdp-43 containing a tev cleavage site. regions of tdp-43 flanking the second rna recognition motif (rrm2) are efficien ...	2011	21454607
exploring the size limit of templates for inhibitors of the m2 ion channel of influenza a virus.	amantadine inhibits the m2 proton channel of influenza a virus, yet its clinical use has been limited by the rapid emergence of amantadine-resistant virus strains. we have synthesized and characterized a series of polycyclic compounds designed as ring-contracted or ring-expanded analogues of amantadine. inhibition of the wild-type (wt) m2 channel and the a/m2-s31n and a/m2-v27a mutant ion channels were measured in xenopus oocytes using two-electrode voltage clamp (tev) assays. several bisnoradam ...	2011	21466220
an intein-cassette integration approach used for the generation of a split tev protease activated by conditional protein splicing.	ligand-induced conditional protein splicing (cps) using a split intein allows the covalent reconstitution of a protein from two polypeptide fragments. the small molecule rapamycin binds to the fused fkbp and frb dimerizer domains and thereby induces folding of the split intein, which then removes itself in the trans-splicing reaction. cps has great potential for the experimental control of protein activity in living cells, however, only one such example was reported yet. this discrepancy is due ...	2011	21487580
hc-pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers.	abstract: background: rna silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. accordingly, plant viruses have evolved to produce counter defensive rna-silencing suppressors (rsss). these factors interfere in various ways with the rna silencing machinery in cells, and thereby disturb the microrna (mirna) mediated endogene regulation and induce developmental and morphological changes in plants. in this study we have explored these effects using ...	2011	21507209
helper component proteinase of genus potyvirus is an interaction partner of translation initiation factors eif(iso)4e and eif4e that contains a 4e binding motif.	the multifunctional helper component proteinase (hcpro) of potyviruses (genus potyvirus; potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eif4e and the isoform eif(iso)4e, which interact with viral genome-linked protein (vpg). here we show that hcpro of potato virus a (pva) interacts with both eif4e and eif(iso)4e, with interactions with eif(is ...	2011	21525344
the variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal.	recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. expression constructs with affinity tags often include an engineered protease site for tag removal. like other enzymes, the activities of proteases can be affected by buffer conditions. the buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. we examined the detergent sensitivity of six commonly-used proteases (enterokinase, f ...	2011	21539919
engineering streptokinase for generation of active site-labeled plasminogen analogs.	we previously demonstrated that streptokinase (sk) can be used to generate active site-labeled fluorescent analogs of plasminogen (pg) by virtue of its nonproteolytic activation of the zymogen. the method is versatile and allows stoichiometric and active site-specific incorporation of any one of many molecular probes. the limitation of the labeling approach is that it is both time-consuming and low yield. here we demonstrate an improved method for the preparation of labeled pg analogs by the use ...	2011	21570944
expression, purification and nmr characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.	the vasopressin type 2 (v2r) receptor belongs to the class of g-protein coupled receptors. it is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. in men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. like most gpcrs, v2r's third intracellular loop (v2r-i3) is involved in the binding and activation of its major effector, the gαs ...	2011	21575724
a tyr residue in the reverse transcriptase domain can mimic the protein-priming tyr residue in the terminal protein domain of a hepadnaviral p protein.	hepadnaviruses are the only known viruses that replicate by protein-primed reverse transcription. beyond the conserved reverse transcriptase (rt) and rnase h domains, their polymerases (p proteins) carry a unique terminal protein (tp) domain that provides a specific tyr-residue, tyr96 in duck hepatitis b virus (dhbv), to which the first nucleotide of minus-strand dna is autocatalytically attached and extended by three more nucleotides. in vitro reconstitution of this priming reaction with dhbv p ...	2011	21593158
[the infection of ixodid ticks collected from humans with the tick-borne encephalitis virus in tomsk city and its suburbs].	the ticks ixodes persulcatus and i. pavlovskyi collected from people visited gardens and suburban forests have been examined by the ifa methods on the presence of tick-borne encephalitis virus (tev). it was established, that the most part of ticks collected from humans belongs to i. persulcatus, despite the fact that i. pavlovskyi dominates on the territory of the city and its suburbs. tev infection was registered more often in fed ticks, in comparison with those without signs of preceding feedi ...	2011	21598663
intracellular reactivation of transcription factors fused with protein transduction domain.	induction of a desired cell type by defined transcription factors (tfs) using ips technology can be used for cell replacement therapy. however, to overcome problems such as tumor formation, genomic insertional mutagenesis by viral transduction in the induction process needs to be avoided using alternative approaches. one approach could be the direct delivery of tf protein by a protein transduction system, whereby a protein transduction domain (ptd) is fused to facilitate the penetration of cell ...	2011	21635926
one is enough: in vivo effective population size is dose-dependent for a plant rna virus.	effective population size (n(e)) determines the strength of genetic drift and the frequency of co-infection by multiple genotypes, making it a key factor in viral evolution. experimental estimates of n(e) for different plant viruses have, however, rendered diverging results. the independent action hypothesis (iah) states that each virion has a probability of infection, and that virions act independent of one another during the infection process. a corollary of iah is that n(e) must be dose depen ...	2011	21750676
is the 6 kda tobacco etch viral protein a bona fide eres marker?	the claim that the 6 kda viral protein (vp) of tobacco etch virus is a marker for er exit sites (eres) has been investigated. when transiently expressed as a cfp tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the copii coat protein yfp-sec24 and the golgi marker man1-rfp. however, when over-expressed the vp locates to larger spherical structures which co-localize with neither er nor golgi markers. nevertheless, deletion of the copi ...	2011	21705387
isolation of transcription factor complexes from arabidopsis cell suspension cultures by tandem affinity purification.	defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. elucidation of the protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. in the last decade, new technologies have emerged to study protein-protein interactions under near-physiological conditions. we have developed a high-throughput tandem affinity p ...	2011	21720954
[biotechnological production of acetylated thymosin beta4].	thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. currently, thymosin beta4 is produced with solid-phase chemical synthesis. biotechnological synthesis of this peptide presents difficulties because n-terminal amino acid residue of thym ...	2011	21721255
super-promoter:tev, a powerful gene expression system for tobacco hairy roots.	in order to identify a promoter system for high-level expression of transgenes in hairy roots, we characterized the chimeric super-promoter fused to the translational enhancer from tobacco etch virus (tev). transgenic tobacco plants and hairy roots were generated with the super-promoter:tev sequence and a modified green fluorescence protein (mgfp5) as a reporter gene. to exploit the utility of hairy root cultures as a secretion-based expression system, the signal peptide of patatin was fused to ...	2012	22160917
expression pattern of recombinant organophosphorus hydrolase from flavobacterium sp. atcc 27551 in escherichia coli.	concerned with the influence of tagging system on the expression of heterogeneous protein in escherichia coli, we attempted to express the organophosphorus hydrolase (oph) of flavobacterium sp. atcc 27551 in e. coli. recombinant oph was overproduced successfully in e. coli when modified without the use of a tobacco etch virus (tev) protease cleavage sequence. in addition, though there has never been a report on the extracellular secretion of recombinant oph harboring native tat signal peptides i ...	2012	23274957
visual tracking of plant virus infection and movement using a reporter myb transcription factor that activates anthocyanin biosynthesis.	insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. their numerous advantages make fluorescent proteins the markers of choice in most studies. however, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. t ...	2012	22238422
rna viral vectors for improved agrobacterium-mediated transient expression of heterologous proteins in nicotiana benthamiana cell suspensions and hairy roots.	plant cell suspensions and hairy root cultures represent scalable protein expression platforms. low protein product titers have thus far limited the application of transient protein expression in these hosts. the objective of this work was to overcome this limitation by harnessing a. tumefaciens to deliver replicating and non-replicating rna viral vectors in plant tissue co-cultures.	2012	22559055
a mitochondrial membrane complex that contains proteins necessary for trna import in trypanosoma brucei.	the mitochondrial genome of trypanosoma brucei does not contain genes encoding trnas, instead this protozoan parasite must import nuclear encoded trnas from the cytosol for mitochondrial translation. previously, it has been shown that mitochondrial trna import requires atp hydrolysis and a proteinaceous mitochondrial membrane component. however, little is known about the mitochondrial membrane proteins involved in trna binding and translocation into the mitochondrion. here we report the purifi ...	2012	22267727
rapid detection of tobacco viruses by reverse transcription loop-mediated isothermal amplification.	tobacco viruses may cause a wide range of diseases that heavily reduce tobacco quality and yield worldwide. in order to detect viral diseases in tobacco fields, a one-step reverse transcription loop-mediated isothermal amplification (rt-lamp) method was established. nucleotide amplification could be observed clearly after adding sybr green i, within 60 min under isothermal conditions, at 63-65 °c with a set of primers targeting the viral coat protein (cp) genes of tobacco viruses including cucum ...	2012	22886186
viruses of pepper crops in the mediterranean basin: a remarkable stasis.	compared to other vegetable crops, the major viral constraints affecting pepper crops in the mediterranean basin have been remarkably stable for the past 20 years. among these viruses, the most prevalent ones are the seed-transmitted tobamoviruses; the aphid-transmitted potato virus y and tobacco etch virus of the genus potyvirus, and cucumber mosaic virus member of the genus cucumovirus; and thrips-transmitted tospoviruses. the last major viral emergence concerns the tospovirus tomato spotted w ...	2012	22682167
a multiplex reverse transcription pcr assay for simultaneous detection of five tobacco viruses in tobacco plants.	tobacco viruses including tobacco mosaic virus (tmv), cucumber mosaic virus (cmv), tobacco etch virus (tev), potato virus y (pvy) and tobacco vein banding mosaic virus (tvbmv) are major viruses infecting tobacco and can cause serious crop losses. a multiplex reverse transcription polymerase chain reaction assay was developed to detect simultaneously and differentiate all five viruses. the system used specific primer sets for each virus producing five distinct fragments 237, 273, 347, 456 and 547 ...	2012	22484613
in vivo and in vitro characterization of tev protease mutants.	tobacco etch virus protease (tevp) is frequently applied in the cleavage of fusion protein. however, production of tev protease in escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type tevp gives rise to the loss-of-function. previously it was reported that tevp s219v displayed more stability, and tevp variant containing t17s/n68d/i77v and double mutant l56v/s135g resulted in the enhanced production and solubility, respectively. here, we introduced t17s/n6 ...	2012	22484199
antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model.	tumstatin, a cleavage fragment of collagen iv, is a potent endogenous inhibitor of angiogenesis. tumstatin-derived peptide t8 possesses all angiostatic properties of full-length tumstatin and indirectly suppresses tumor growth. the potential of t8 to block pathological angiogenesis in the eye has not been explored yet. here we assess antiangiogenic effects of a recombinant t8 peptide in rabbit corneal neovascularization models. the fusion protein consisting of t8 and thioredoxin was synthesized ...	2012	22440655
recombinant production of the therapeutic peptide lunasin.	lunasin is a chemopreventive peptide produced in a number of plant species. it comprises a helical region with homology to a region of chromatin binding proteins, an arg-gly-asp cell adhesion motif and eight aspartic acid residues. in vitro studies indicate that lunasin suppresses chemical and oncogene driven transformation of mammalian cells. we have explored efficient recombinant production of lunasin by exploiting the clostridium thermocellum cipb cellulose binding domain (cbd) as a fusion pa ...	2012	22376274
functional cell surface display and controlled secretion of diverse agarolytic enzymes by escherichia coli with a novel ligation-independent cloning vector based on the autotransporter yfal.	autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. we adopted an autotransporter (yfal) of escherichia coli for the cell surface display system. the critical regions in yfal for surface display were identified for the construction of a ligation-independent cloning (lic)-based display system. the designed system showed no detrimental effect on either the growth of the host cell or ...	2012	22344647
tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives.	tobacco etch virus (tev) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. it is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of tev protease. consequently, tedious desalination or the substitution of standard tev reaction buffer for elution buffer are often needed to ensure tev protease activity when r ...	2012	22285121
identification of protein complexes in escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.	since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (ppi) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems(1, 2). physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity ...	2012	23168686
expression and purification of sfax(ii), a protein involved in regulating adhesion and motility genes in extraintestinal pathogenic escherichia coli.	pathogenic escherichia coli strains commonly harbor genes involved in formation of fimbriae, such as the sfa(ii) fimbrial gene cluster found in uropathogenic and newborn meningitis isolates. the sfax(ii) gene, located at the distal end of the sfa(ii) operon, was recently shown to play a role in controlling virulence-related gene expression in extraintestinal pathogenic e. coli (expec). until now, detailed characterization of the sfax(ii) protein has been hampered by difficulties in obtaining lar ...	2012	23022032
efficient expression and purification of tag-free epstein-barr virus ebna1 protein in escherichia coli by auto-induction.	epstein-barr nuclear antigen 1 (ebna1) is the essential epstein-barr virus (ebv) protein at the interface between the ebv genome and the host chromatin. it is ebna1's task to guarantee replication and segregation of the multicopy closed circular viral genome in infected cells. while ebna1's functions are relatively well understood, little is known about the molecular mechanisms of ebna1 mediating chromatin tethering and dna replication. to characterize those, purified ebna1 would be a very usefu ...	2012	22944205
the rtm resistance to potyviruses in arabidopsis thaliana: natural variation of the rtm genes and evidence for the implication of additional genes.	the non conventional rtm (restricted tobacco etch virus movement) resistance which restricts long distance movement of some plant viruses in arabidopsis thaliana is still poorly understood. though at least three rtm genes have been identified, their precise role(s) in the process as well as whether other genes are involved needs to be elucidated.	2012	22723957
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
identification of protein interacting partners using tandem affinity purification.	a critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. there are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). recent studies have highlighted the utility of double-affinity t ...	2012	22395237
tobacco etch virus infectivity in capsicum spp. is determined by a maximum of three amino acids in the viral virulence determinant vpg.	potyvirus resistance in capsicum spp. has been attributed to amino acid substitutions at the pvr1 locus that cause conformational shifts in eukaryotic translation initiation factor eif4e. the viral genome-linked protein (vpg) sequence was isolated and compared from three tobacco etch virus (tev) strains, highly aphid-transmissible (hat), mex21, and n, which differentially infect capsicum genotypes encoding pvr1(+), pvr1, and pvr1(2). viral chimeras were synthesized using the tev-hat genome, repl ...	2012	23134519
inhibition of pokeweed antiviral protein (pap) by turnip mosaic virus genome-linked protein (vpg).	pokeweed antiviral protein (pap) from phytolacca americana is a ribosome-inactivating protein (rip) and an rna n-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rrna, arresting protein synthesis at the translocation step. pap is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. to elucidate the mechanism of rna depurination, and to understand how pap recognizes and targets various rnas, the interactions ...	2012	22773840
effects of potyvirus effective population size in inoculated leaves on viral accumulation and the onset of symptoms.	effective population size (n(e)) is a key parameter for understanding evolutionary processes, but it is generally not considered in epidemiological studies or in studying infections of individual hosts. whether n(e) has an effect on the onset of symptoms and viral accumulation in tobacco etch virus (tev) infection of nicotiana tabacum plants is considered here. using mixtures of tev variants carrying fluorescent markers, the dose dependence of n(e) was confirmed, and the inoculation procedure wa ...	2012	22740417
transcript profiling of different arabidopsis thaliana ecotypes in response to tobacco etch potyvirus infection.	the use of high-throughput transcript profiling techniques has opened the possibility of identifying, in a single experiment, multiple host mrnas whose levels of accumulation are altered in response to virus infection. several studies have used this approach to analyze the response of arabidopsis thaliana to the infection by different rna and dna viruses. however, the possible differences in response of genetically heterogeneous ecotypes of the plant to the same virus have never been addressed b ...	2012	22737149
magnitude and sign epistasis among deleterious mutations in a positive-sense plant rna virus.	how epistatic interactions between mutations determine the genetic architecture of fitness is of central importance in evolution. the study of epistasis is particularly interesting for rna viruses because of their genomic compactness, lack of genetic redundancy, and apparent low complexity. moreover, interactions between mutations in viral genomes determine traits such as resistance to antiviral drugs, virulence and host range. in this study we generated 53 tobacco etch potyvirus genotypes carry ...	2012	22491062
type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis.	apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. one of them, the type 1 inositol-1,4,5-trisphosphate receptor (ip(3) r-1), a multimeric receptor located on the endoplasmic reticulum (er) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (sts) induced-apoptosis in jurkat cells. because the reported cleavage site separates the ip(3) binding site from the channel moiety, its cleavage would shut down a critical signaling ...	2012	22473799
immune response to a potyvirus with exposed amino groups available for chemical conjugation.	the amino terminus of the tobacco etch virus (tev) capsid protein is located on the external surface of infectious tev particles, as proposed by previous studies and an in silico model. the epsilon amino groups on the exposed lysine residues are available for chemical conjugation to any given protein, and can thus act as antigen carriers. the availability of amino groups on the surfaces of tev particles was determined and the immune response to tev evaluated.	2012	22452850
tipi: tev protease-mediated induction of protein instability.	reverse genetics approaches require methods to inactivate a specific protein. one possibility is to modify the target protein with a degradation signal (degron). degrons are short, transferable sequences that confer protein instability. they target proteins for degradation either constitutively or after activation, e.g., by phosphorylation, presence of a binding partner, or conformational rearrangements in the substrate. in this chapter, we describe a synthetic way to activate a degron. it emplo ...	2012	22350916
a modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells.	despite progress in the development of methods to monitor protein interactions, studies of interactions between membrane proteins in mammalian cells remain challenging. protein complementation assays (pcas) are commonly used to study interactions between proteins due to their simplicity. they are based on interaction-mediated reconstitution of a reporter protein, which can be easily monitored. recently, a protein complementation method named split-tev (tobacco etch virus) has been developed and ...	2012	22342621
lectin-mediated resistance impairs plant virus infection at the cellular level.	plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. this study analyzed the resistance of certain arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (plamv). map-based positional cloning revealed that the lectin gene jacalin-type lectin required for p ...	2012	22307853
oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins.	the tobacco etch virus (tev) protease is a useful tool for the removal of fusion tags from recombinant proteins. the difficulty in obtaining this enzyme led us to look for an optimal method for its use. in this work, we produced both the wild-type and the s219v mutant tev proteases fused to the streptag ii affinity sequence (streptag ii-tev(wt), and streptag ii-tev(s219v), respectively). the two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective f ...	2012	22300512
intracellular detection and evolution of site-specific proteases using a genetic selection system.	development of endoproteases, programmed to promote degradation of peptides or proteins responsible for pathogenic states, represents an attractive therapeutic strategy, since such biocatalytic agents could be directed against a potentially unlimited repertoire of extracellular proteinaceous targets. difficulties associated with engineering enzymes with tailor-made substrate specificities have, however, hindered the discovery of proteases possessing both the efficiency and selectivity to act as ...	2012	22270548
several affinity tags commonly used in chromatographic purification.	affinity tags have become powerful tools from basic biological research to structural and functional proteomics. they were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, flag tag, strep ii tag, streptavidin-binding peptide (sbp) tag, calmodulin-binding peptide (cbp), glutathione s-tr ...	2013	24490106
crystal structures of phanerochaete chrysosporium pyranose 2-oxidase suggest that the n-terminus acts as a propeptide that assists in homotetramer assembly.	the flavin-dependent homotetrameric enzyme pyranose 2-oxidase (p2o) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β-d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. here, we present crystal structures of p2o from the efficient lignocellulolytic basidiomycete phanerochaete chrysosporium. structures were determined of wild-type pcp2o from the natural fungal source, ...	2013	24282677
preferential apelin-13 production by the proprotein convertase pcsk3 is implicated in obesity.	the peptide hormone apelin is translated as a 77-residue preproprotein, truncated to the 55-residue proapelin and, subsequently, to 13-36-residue bioactive isoforms named apelin-13 to -36. proapelin is hypothesized to be cleaved to apelin-36 and then to the shorter isoforms. however, neither the mechanism of proapelin processing nor the endoproteases involved have been determined. we show direct cleavage of proapelin to apelin-13 by proprotein convertase subtilisin/kexin 3 (pcsk3, or furin) in v ...	2013	24251091
stability and fitness impact of the visually discernible rosea1 marker in the tobacco etch virus genome.	antirrhinum majus rosea1 (ros1) is an myb-related transcription factor that induces anthocyanin biosynthesis in plant tissues, and has been shown to be suitable for visual tracking of virus infection in plants. however, activation of anthocyanin biosynthesis has far reaching effects on plant physiology and could consequently have negative effects on viral replication. therefore, viruses carrying the ros1 marker might have a low fitness and consequently rapidly lose the marker. to compare the sta ...	2013	24022073
crystal structure of the effector protein xoo4466 from xanthomonas oryzae.	many gram-negative bacteria deliver their virulence factors into host cells through a secretion system. those factors, called effector proteins, are involved in the pathogenicity in host cells by interfering with various cellular events. the phytopathogen xanthomonas oryzae pv. oryzae uses a type iii secretion system to inject its effectors, but the functional roles of these proteins remain largely uncharacterized. here, we determined a crystal structure of xoo4466, an effector from x. oryzae pv ...	2013	24007778
interaction of mouse ttc30/dyf-1 with multiple intraflagellar transport complex b proteins and kif17.	intraflagellar transport (ift) is a microtubule based system that supports the assembly and maintenance of cilia. genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the ift machinery. in this study we prepared mouse pituitary cells that expressed an epitope-tagged ift protein and immuno-purified the ift b complex from these cells. mass spectrometry analysis of the isolated complex led to identification of a number of well known co ...	2013	23810713
crystal structures of the first condensation domain of cda synthetase suggest conformational changes during the synthetic cycle of nonribosomal peptide synthetases.	nonribosomal peptide synthetases (nrpss) are large modular macromolecular machines that produce small peptide molecules with wide-ranging biological activities, such as antibiotics and green chemicals. the condensation (c) domain is responsible for amide bond formation, the central chemical step in nonribosomal peptide synthesis. here we present two crystal structures of the first condensation domain of the calcium-dependent antibiotic (cda) synthetase (cda-c1) from streptomyces coelicolor, dete ...	2013	23756159
insights into notch3 activation and inhibition mediated by antibodies directed against its negative regulatory region.	notch receptors are single-pass transmembrane proteins that regulate development and tissue homeostasis in all metazoan organisms. prior to ligand-induced signaling, notch receptors adopt a proteolytic resistant conformation maintained by a critical interdomain interface within a negative regulatory region (nrr), which sits immediately external to the plasma membrane. signaling is initiated when ligand binding induces exposure of the proteolytic cleavage site, termed s2, within the nrr. here, we ...	2013	23747483
genotypic but not phenotypic historical contingency revealed by viral experimental evolution.	the importance of historical contingency in determining the potential of viral populations to evolve has been largely unappreciated. identifying the constraints imposed by past adaptations is, however, of importance for understanding many questions in evolutionary biology, such as the evolution of host usage dynamics by multi-host viruses or the emergence of escape mutants that persist in the absence of antiviral treatments. to address this issue, we undertook an experimental approach in which s ...	2013	23421472
differential temperature dependence of tobacco etch virus and rhinovirus 3c proteases.	because of their stringent sequence specificity, the 3c-like proteases from tobacco etch virus (tev) and human rhinovirus are often used for the removal of affinity tags. the latter enzyme is rumored to have greater catalytic activity at 4 °c, the temperature at which fusion protein substrates are usually digested. here we report that experiments with fusion protein and peptide substrates confirm this conjecture. whereas the catalytic efficiency of rhinovirus 3c protease is approximately the sam ...	2013	23395976
saccharomyces cerevisiae-based platform for rapid production and evaluation of eukaryotic nutrient transporters and transceptors for biochemical studies and crystallography.	to produce large quantities of high quality eukaryotic membrane proteins in saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins c-terminally-fused to a tobacco etch virus (tev) protease detachable green fluorescent protein (gfp)-8his tag, which facilitates localization, quantification, quality control, and purification. using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from s. cer ...	2013	24124599
a tobacco etch virus protease with increased substrate tolerance at the p1' position.	site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. they are widely used for diverse applications, like protein purification, assessment of protein-protein interactions or regulation of protein localization, abundance or activity. here, we report the development of a procedure to select protease variants with altered specificity based on the well-established saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. we applied this method ...	2013	23826349
the dead-box protein dbp2 functions with the rna-binding protein yra1 to promote mrnp assembly.	eukaryotic gene expression involves numerous biochemical steps that are dependent on rna structure and ribonucleoprotein (rnp) complex formation. the dead-box class of rna helicases plays fundamental roles in formation of rna and rnp structure in every aspect of rna metabolism. in an effort to explore the diversity of biological roles for dead-box proteins, our laboratory recently demonstrated that the dead-box protein dbp2 associates with actively transcribing genes and is required for normal g ...	2013	23721653
development of multiplex real-time pcr for simultaneous detection of three potyviruses in tobacco plants.	to develop a multiplex real-time pcr assay using taqman probes for the simultaneous detection and quantification of tobacco etch virus (tev), potato virus y (pvy) and tobacco vein banding mosaic virus (tvbmv).	2013	23164070
the absence of eukaryotic initiation factor eif(iso)4e affects the systemic spread of a tobacco etch virus isolate in arabidopsis thaliana.	translation initiation factor eif4e exerts an important role during infection of viral species in the family potyviridae. particularly, a eif(iso)4e family member is required for arabidopsis thaliana susceptibility to turnip mosaic virus, lettuce mosaic virus, and tobacco etch virus (tev). in addition, a resistance mechanism named restriction of tev movement (rtm) in a. thaliana controls the systemic spread of tev in col-0 ecotype. here, we describe that tev-tamps, a mexican isolate, overcomes t ...	2013	23252462
mutation of a short variable region in hcpro protein of potato virus a affects interactions with a microtubule-associated protein and induces necrotic responses in tobacco.	helper component proteinase (hcpro) is a multifunctional protein of potyviruses (genus potyvirus). hcpro of potato virus a (pva) interacts with the microtubule-associated protein hip2 in host cells, and depletion of hip2 reduces virus accumulation. this study shows that hcpro of potato virus y and tobacco etch virus also interact with hip2. the c-proximal portion of pva hcpro determines the interaction with hip2 and was found to contain a stretch of six residues comprising a highly variable regi ...	2013	23514111
effects of thioredoxin: sumo and intein on soluble fusion expression of an antimicrobial peptide og2 in escherichia coli.	og2 is a modified antimicrobial peptide of palustrin-og1 (og1), which is derived from odorrana grahami frog. og2 has shown much higher selective antimicrobial activity and lower hemolytic activity than og1, indicating og2 may be a promising antimicrobial agent. in this study, we investigated three fusion partners, including thioredoxin, mxe gyra intein, and small ubiquitin-like modifier (sumo), each fused with og2, and examined their effects on the expression level and solubility of og2 in esche ...	2013	22670762
engineering of tev protease variants by yeast er sequestration screening (yess) of combinatorial libraries.	myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (er). a substrate fusion protein composed of yeast adhesion receptor subunit aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a c-term ...	2013	23589865
chic, a new tandem affinity tag for the protein purification toolbox.	in the present work we have constructed a new tandem affinity purification tag and used it to purify two different polypeptides, pcsb and ecl1 from streptococcus pneumoniae. pcsb probably functions as a peptidoglycan hydrolase and is believed to be involved in splitting of the septum during cell division. ecl1 is the extracellular domain of the membrane spanning protein ftsx. experimental evidence indicates that the ecl1 domain controls the activity of pcsb through direct interaction (sham et al ...	2013	23154041
development of a 2,4-dinitrotoluene-responsive synthetic riboswitch in e. coli cells.	riboswitches are rna sequences that regulate expression of associated downstream genes in response to the presence or absence of specific small molecules. a novel riboswitch that activates protein translation in e. coli cells in response to 2,4-dinitrotoluene (dnt) has been engineered. a plasmid library was constructed by incorporation of 30 degenerate bases between a previously described trinitrotoluene aptamer and the ribosome binding site. screening was performed by placing the riboswitch lib ...	2013	23092157
cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs.	a membrane-embedded curdlan synthase (crds) from agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from udp-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. we report wheat germ cell-free protein synthesis (wg-cfps) of full-length crds containing a 6xhis affinity tag and either factor xa or tobacco etch virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. full-length crds was synthesised with no variations in primary st ...	2013	23063656
role of the viral hemagglutinin in the anti-influenza virus activity of newly synthesized polycyclic amine compounds.	we here report on the synthesis of new series of polycyclic amines initially designed as ring-rearranged analogs of amantadine and featuring pentacyclo, hexacyclo, and octacyclo rings. a secondary amine, 3-azahexacyclo[7.6.0.0¹,⁵.0⁵,¹².0⁶,¹⁰.0¹¹,¹⁵]pentadeca-7,13-diene, 3, effectively inhibited a/m2 proton channel function, and, moreover, possessed dual activity against an a/h3n2 virus carrying a wild-type a/m2 proton channel, as well as an amantadine-resistant a/h1n1 virus. among the polycyclic ...	2013	23800838
conservation and variability in the structure and function of the cas5d endoribonuclease in the crispr-mediated microbial immune system.	clustered regularly interspaced short palindromic repeats (crisprs) and crispr-associated (cas) proteins form an rna-mediated microbial immune system against invading foreign genetic elements. cas5 proteins constitute one of the most prevalent cas protein families in crispr-cas systems and are predicted to have rna recognition motif (rrm) domains. cas5d is a subtype i-c-specific cas5 protein that can be divided into two distinct subgroups, one of which has extra c-terminal residues while the oth ...	2013	23500492
high prevalence of poleroviruses in field-grown pepper in turkey and tunisia.	open-field pepper crops were sampled in 2011 in turkey and tunisia and surveyed for the major pepper-infecting viruses. as expected, potato virus y and cucumber mosaic virus (in both countries), and tobacco etch virus (in turkey only) were quite frequent. however, poleroviruses were the most common viruses, with prevalences above 70 %. partial sequence analyses revealed the occurrence of poleroviruses resembling either beet western yellows virus (bwyv) or pepper vein yellows virus in the sampled ...	2013	23183831
milligram quantities of homogeneous recombinant full-length mouse munc18c from escherichia coli cultures.	vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. the sec/munc18 protein munc18c is essential for insulin-regulated trafficking of glucose transporter4 (glut4) vesicles to the cell surface in muscle and adipose tissue. previously, our biophysical and structural studies have used munc18c expressed in sf9 insect cells. however to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of munc18c, we investigat ...	2013	24391775
insight into the structural stability of wild type and mutants of the tobacco etch virus protease with molecular dynamics simulations.	the efficiency and high specificity of tobacco etch virus protease (tevp) has made it widely used for cleavage of recombinant fusion proteins. however, tevp suffers from a few intrinsic defects such as self-cleavage, poorly expressed in e. coli and less soluble. so some mutants were designed to improve it, such as s219v, t17s/n68d/i77v and l56v/s135g etc. md simulations for the wt tevp and its mutants were performed to explore the underlying dynamic effects of mutations on tevp. although the glo ...	2013	24043540
engineering soluble tobacco etch virus protease accompanies the loss of stability.	tobacco etch virus protease (tevp) is a widely used tool enzyme in biological studies. to improve the solubility of recombinant tevp, three variants, including the double mutant (l56v/s135g), the triple mutant (t17s/n68d/i77v), and the quintuple mutant (t17s/l56v/n68d/i77v/s135g), have been developed, however, with little information on functional stability. here we investigated the solubility and stability of the three tevp mutants under different temperature and denaturants, and in escherichia ...	2013	24012464
the novel structure of the cockroach allergen bla g 1 has implications for allergenicity and exposure assessment.	sensitization to cockroach allergens is a major risk factor for asthma. the cockroach allergen bla g 1 has multiple repeats of approximately 100 amino acids, but the fold of the protein and its biological function are unknown.	2013	23915714
distinct regions of the escherichia coli parc c-terminal domain are required for substrate discrimination by topoisomerase iv.	type iia dna topoisomerases are essential enzymes that use atp to maintain chromosome supercoiling and remove links between sister chromosomes. in escherichia coli, the type iia topoisomerase topo iv rapidly removes positive supercoils and catenanes from dna but is significantly slower when confronted with negatively supercoiled substrates. the ability of topo iv to discriminate between positively and negatively supercoiled dna requires the c-terminal domain (ctd) of one of its two subunits, par ...	2013	23867279
expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor cb2.	human peripheral cannabinoid receptor cb2, a g protein-coupled receptor (gpcr) involved in regulation of immune response has become an important target for pharmaceutical drug development. structural and functional studies on cb2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. the goal of this project was to develop a generic strategy for preparation of functional recombinant cb2 and ...	2013	23777860
application of halotag technology to expression and purification of cannabinoid receptor cb2.	expression of milligram quantities of functional, stable g protein-coupled receptors (gpcr) for high-resolution structural studies remains a challenging task. the goal of this work was to evaluate the usefulness of the halotag system (promega) for expression and purification of the human cannabinoid receptor cb(2), an important target for development of drugs for treatment of immune disorders, inflammation, and pain. here we investigated expression in escherichia coli cells of the integral membr ...	2013	23470778
3-azatetracyclo[5.2.1.1(5,8).0(1,5)]undecane derivatives: from wild-type inhibitors of the m2 ion channel of influenza a virus to derivatives with potent activity against the v27a mutant.	we have synthesized and characterized a series of compounds containing the 3-azatetracyclo[5.2.1.1(5,8).0(1,5)]undecane scaffold designed as analogues of amantadine, an inhibitor of the m2 proton channel of influenza a virus. inhibition of the wild-type (wt) m2 channel and the amantadine-resistant a/m2-s31n and a/m2-v27a mutant ion channels were measured in xenopus oocytes using two-electrode voltage clamp (tev) assays. most of the novel compounds inhibited the wt ion channel in the low micromol ...	2013	24237039
do-it-yourself histidine-tagged bovine enterokinase: a handy member of the protein engineer's toolbox.	enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its n-terminal propeptide. due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate n-terminal affinity tags from target proteins with authentic n-termini. in order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase ligh ...	2013	24184090
structural determinants of oligomerization of δ(1)-pyrroline-5-carboxylate dehydrogenase: identification of a hexamerization hot spot.	the aldehyde dehydrogenase (aldh) superfamily member δ(1)-pyrroline-5-carboxylate dehydrogenase (p5cdh) catalyzes the nad(+)-dependent oxidation of glutamate semialdehyde to glutamate, which is the final step of proline catabolism. defects in p5cdh activity lead to the metabolic disorder type ii hyperprolinemia, p5cdh is essential for virulence of the fungal pathogen cryptococcus neoformans, and bacterial p5cdhs have been targeted for vaccine development. although the enzyme oligomeric state is ...	2013	23747974
identification and characterization of a mucilaginibacter sp. strain qm49 β-glucosidase and its use in the production of the pharmaceutically active minor ginsenosides (s)-rh1 and (s)-rg2.	here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (bglqm) from mucilaginibacter sp. strain qm49 that shows biotransformation activity for various major ginsenosides. the gene responsible for this activity, bglqm, consists of 2,346 bp and is predicted to encode 781 amino acid residues. this enzyme has a molecular mass of 85.6 kda. sequence analysis of bglqm revealed that it could be classified into glycoside hydrolase family 3. the enzyme was overexpressed in escher ...	2013	23811513
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
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
recombinant production of the amino terminal cytoplasmic region of dengue virus non-structural protein 4a for structural studies.	dengue virus (denv) is a mosquito-transmitted positive single strand rna virus belonging to the flaviviridae family. denv causes dengue fever, currently the world's fastest-spreading tropical disease. severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. there is no specific treatment and no anti-denv vaccines. our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4a (ns4a) comprising ami ...	2014	24466115
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
investigating the interactions of the 18kda translocator protein and its ligand pk11195 in planar lipid bilayers.	the functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. we have investigated the interaction of a drug ligand, pk11195, with its primary protein target, the integral membrane 18kda translocator protein (tspo), and model membranes using langmuir monolayers, quartz crystal microbalance with dissipation monitoring (qcm-d) and neutron reflectometry (nr). we found that pk11195 is incorporated into li ...	2014	24374318
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
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
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
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
recombinant expression, purification and characterization of antimicrobial peptide orbk in escherichia coli.	orbk (lkgcwtksippkpcfk) is a cyclic cationic peptide that has potent antimicrobial properties and trypsin inhibitory activities. to explore a new approach for expressing orbk in escherichia coli, a sequence encoding orbk was cloned into pet28a vector in which maltose-binding protein (mbp) was used as a fusion partner and an n-terminal 6-his as an affinity tag. protein expression was induced with 0.5mm isopropyl-thio-galactoside (iptg) for 4h at 37°c. the recombinant orbk was then purified by ni ...	2014	24398234
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