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1.
Raffaele Lombardi Patrizia Circelli Maria Elena Villani Giampaolo Buriani Luca Nardi Valentina Coppola Linda Bianco Eugenio Benvenuto Marcello Donini Carla Marusic 《BMC biotechnology》2009,9(1):96
Background
In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. 相似文献2.
3.
Phytophthora infestans INF1 elicitin causes the hypersensitive response (HR) in Nicotiana benthamiana (Kamoun et al. in Plant Cell 10:1413–1425, 1998). To identify N. benthamiana proteins that interact with INF1, we carried out a yeast two-hybrid screen. This screen resulted in the isolation of a gene
NbLRK1 coding for a novel lectin-like receptor kinase. NbLRK1 interacted with INF1 through its VIb kinase subdomain. Purified INF1
and NbLRK1 proteins also interacted in vitro. INF1 treatment of N. benthamiana leaves induced autophosphorylation of NbLRK1. Most importantly, virus-induced gene silencing (VIGS) of NbLRK1 delayed INF1-mediated HR in N. benthamiana. These data suggest that NbLRK1 is a component of the N. benthamiana protein complex that recognizes INF1 elicitor and transduces the HR signal. 相似文献
4.
Dissecting virulence function from recognition: cell death suppression in Nicotiana benthamiana by XopQ/HopQ1‐family effectors relies on EDS1‐dependent immunity
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Many Gram‐negative plant pathogenic bacteria express effector proteins of the XopQ/HopQ1 family which are translocated into plant cells via the type III secretion system during infection. In Nicotiana benthamiana, recognition of XopQ/HopQ1 proteins induces an effector‐triggered immunity (ETI) reaction which is not associated with strong cell death but renders plants immune against Pseudomonas syringae and Xanthomonas campestris pv. vesicatoria strains. Additionally, XopQ suppresses cell death in N. benthamiana when transiently co‐expressed with cell death inducers. Here, we show that representative XopQ/HopQ1 proteins are recognized similarly, likely by a single resistance protein of the TIR‐NB‐LRR class. Extensive analysis of XopQ derivatives indicates the recognition of structural features. We performed Agrobacterium‐mediated protein expression experiments in wild‐type and EDS1‐deficient (eds1) N. benthamiana leaves, not recognizing XopQ/HopQ1. XopQ recognition limits multiplication of Agrobacterium and attenuates levels of transiently expressed proteins. Remarkably, XopQ fails to suppress cell death reactions induced by different effectors in eds1 plants. We conclude that XopQ‐mediated cell death suppression in N. benthamiana is due to the attenuation of Agrobacterium‐mediated protein expression rather than the cause of the genuine XopQ virulence activity. Thus, our study expands our understanding of XopQ recognition and function, and also challenges the commonly used co‐expression assays for elucidation of in planta effector activities, at least under conditions of ETI induction. 相似文献
5.
Xue Yang Yanzhen Tian Xing Zhao Liangliang Jiang Ying Chen Shuzhen Hu Stuart MacFarlane Jianping Chen Yuwen Lu Fei Yan 《Molecular Plant Pathology》2020,21(11):1495-1501
Systemic necrosis often occurs during viral infection of plants and is thought mainly to be the result of long-term stress induced by viral infection. Potato virus X (PVX) encodes the P25 pathogenicity factor that triggers a necrotic reaction during PVX-potato virus Ysynergistic coinfection. In this study, we discovered that NbALY916, a multifunctional nuclear protein, could interact with P25. When NbALY916 expression was reduced by tobacco rattle virus (TRV)-based virus-induced gene silencing, the accumulation of P25 was increased, which would be expected to cause more severe necrosis. However, silencing of NbALY916 reduced the extent of cell death caused by P25. Furthermore, we found that overexpression of NbALY916 increased the accumulation of H2O2 and triggered more extensive cell death when coexpressed with P25, even though accumulation of P25 was itself reduced by the increased expression of NbALY916. Furthermore, transient expression of P25 specifically induced the expression of NbALY916 mRNA, but not the mRNAs of three other ALYs in Nicotiana benthamiana. In addition, we showed that silencing of NbALY916 or transient overexpression of NbALY916 affected the infection of PVX in N. benthamiana. Our results reveal that NbALY916 has an antiviral role that, in the case of PVX, operates by inducing the accumulation of H2O2 and mediating the degradation of P25. 相似文献
6.
Benjamin B. Gengenbach Linda L. Keil Patrick Opdensteinen Catherine R. Müschen Georg Melmer Hans Lentzen Jens Bührmann Johannes F. Buyel 《Biotechnology and bioengineering》2019,116(9):2236-2249
Cancer is the leading cause of death in industrialized countries. Cancer therapy often involves monoclonal antibodies or small-molecule drugs, but carbohydrate-binding lectins such as mistletoe (Viscum album) viscumin offer a potential alternative treatment strategy. Viscumin is toxic in mammalian cells, ruling them out as an efficient production system, and it forms inclusion bodies in Escherichia coli such that purification requires complex and lengthy refolding steps. We therefore investigated the transient expression of viscumin in intact Nicotiana benthamiana plants and Nicotiana tabacum Bright Yellow 2 plant-cell packs (PCPs), comparing a full-length viscumin gene construct to separate constructs for the A and B chains. As determined by capillary electrophoresis the maximum yield of purified heterodimeric viscumin in N. benthamiana was ~7 mg/kg fresh biomass with the full-length construct. The yield was about 50% higher in PCPs but reduced 10-fold when coexpressing A and B chains as individual polypeptides. Using a single-step lactosyl-Sepharose affinity resin, we purified viscumin to ~54%. The absence of refolding steps resulted in estimated cost savings of more than 80% when transient expression in tobacco was compared with E. coli. Furthermore, the plant-derived product was ~3-fold more toxic than the bacterially produced counterpart. We conclude that plants offer a suitable alternative for the production of complex biopharmaceutical proteins that are toxic to mammalian cells and that form inclusion bodies in bacteria. 相似文献
7.
We have developed Cucumber mosaic virus (CMV) as a plant virus vector especially for production of pharmaceutical proteins. The CMV vector is a vector modifiable
for different host plants and does not require further engineering steps. CMV contains three genomic RNA molecules (RNAs 1–3)
necessary for infectivity. With this system, instead of creating different vector constructs for each plant we use, we take
advantage of the formation of pseudrecombinants between two CMV isolates by simply reassembling a vector construct (RNA 2
base) and an RNA molecule containing the host determinant (mostly RNA 3). In this study, the gene for acidic fibroblast growth
factor (aFGF), one of the human cytokines, was cloned under the control of the subgenomic promoter for RNA 4A of the CMV-based
vector, C2-H1. Infected Nicotiana benthamiana plants produced aFGF at levels up to 5–8% of the total soluble protein. The tobacco-produced aFGF was purified, and its biological
activity was confirmed. Using this system, which provides a versatile and viable strategy for the production of therapeutic
proteins in plants, we also demonstrated a high level of aFGF in Glycine max (soybean) and Arabidopsis thaliana. 相似文献
8.
Claudia Hackenberg Annerose Engelhardt Hans C. P. Matthijs Floyd Wittink Hermann Bauwe Aaron Kaplan Martin Hagemann 《Planta》2009,230(4):625-637
In cyanobacteria, photorespiratory 2-phosphoglycolate (2PG) metabolism is mediated by three different routes, including one
route involving the glycine decarboxylase complex (Gcv). It has been suggested that, in addition to conversion of 2PG into
non-toxic intermediates, this pathway is important for acclimation to high-light. The photoreduction of O2 (Mehler reaction), which is mediated by two flavoproteins Flv1 and Flv3 in cyanobacteria, dissipates excess reductants under
high-light by the four electron-reduction of oxygen to water. Single and double mutants defective in these processes were
constructed to investigate the relation between photorespiratory 2PG-metabolism and the photoreduction of O2 in the cyanobacterium Synechocystis sp. PCC 6803. The single mutants Δflv1, Δflv3, and ΔgcvT, as well as the double mutant Δflv1/ΔgcvT, were completely segregated but not the double mutant Δflv3/ΔgcvT, suggesting that the T-protein subunit of the Gcv (GcvT) and Flv3 proteins cooperate in an essential process. This assumption
is supported by the following results: (1) The mutant Δflv3/ΔgcvT showed a considerable longer lag phase and sometimes bleached after shifts from slow (low light, air CO2) to rapid (standard light, 5% CO2) growing conditions. (2) Photoinhibition experiments indicated a decreased ability of the mutant Δflv3/ΔgcvT to cope with high-light. (3) Fluorescence measurements showed that the photosynthetic electron chain is reduced in this mutant.
Our data suggest that the photorespiratory 2PG-metabolism and the photoreduction of O2, particularly that catalyzed by Flv3, cooperate during acclimation to high-light stress in cyanobacteria.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
9.
Comparison of VHH‐Fc antibody production in Arabidopsis thaliana,Nicotiana benthamiana and Pichia pastoris
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Thomas De Meyer Bram Laukens Jonah Nolf Els Van Lerberge Riet De Rycke Ans De Beuckelaer Sylvie De Buck Nico Callewaert Ann Depicker 《Plant biotechnology journal》2015,13(7):938-947
VHHs or nanobodies are widely acknowledged as interesting diagnostic and therapeutic tools. However, for some applications, multivalent antibody formats, such as the dimeric VHH‐Fc format, are desired to increase the functional affinity. The scope of this study was to compare transient expression of diagnostic VHH‐Fc antibodies in Nicotiana benthamiana leaves with their stable expression in Arabidopsis thaliana seeds and Pichia pastoris. To this end, VHH‐Fc antibodies targeting green fluorescent protein or the A. thaliana seed storage proteins (albumin and globulin) were produced in the three platforms. Differences were mainly observed in the accumulation levels and glycosylation patterns. Interestingly, although in plants oligomannosidic N‐glycans were expected for KDEL‐tagged VHH‐Fcs, several VHH‐Fcs with an intact KDEL‐tag carried complex‐type N‐glycans, suggesting a dysfunctional retention in the endoplasmic reticulum. All VHH‐Fcs were equally functional across expression platforms and several outperformed their corresponding VHH in terms of sensitivity in ELISA. 相似文献
10.
Julia Jansing Markus Sack Sruthy Maria Augustine Rainer Fischer Luisa Bortesi 《Plant biotechnology journal》2019,17(2):350-361
Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N‐linked glycans, including the presence of β‐1,2‐xylose and core α‐1,3‐fucose residues in plants, can affect the activity, potency and immunogenicity of plant‐derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N‐glycosylation machinery to allow the synthesis of complex N‐glycans lacking β‐1,2‐xylose and core α‐1,3‐fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant‐specific α‐1,3‐fucosyltransferase and β‐1,2‐xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry‐based N‐glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64‐binding affinity of 2G12 glycovariants produced in wild‐type N. benthamiana, the newly generated FX‐KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco‐engineered antibody performed as well as its CHO‐produced counterpart. 相似文献
11.
Loeffler M Le'Negrate G Krajewska M Reed JC 《Cancer immunology, immunotherapy : CII》2009,58(5):769-775
Intravenously-applied bacteria tend to accumulate in tumors and can sporadically lead to tumor regression. Systemic administration
of attenuated Salmonella typhimurium is safe and has shown no significant adverse effects in humans. The purpose of this study was to test the hypothesis that
engineering S. typhimurium to express a chemokine, CCL21, would increase anti-tumor activity. We engineered an attenuated strain of S. typhimurium to produce the chemokine CCL21. Attenuated S. typhimurium expressing CCL21 significantly inhibited the growth of primary tumors and pulmonary metastases in preclinical models of multi-drug-resistant
murine carcinomas, while control bacteria did not. Histological analysis of tumors showed marked inflammatory cell infiltrates
in mice treated with CCL21-expressing but not control bacteria. Levels of cytokines and chemokines known to be induced by
CCL21 [e.g., interferon-γ (INFγ), CXCL9, and CXCL10] were significantly elevated in tumors of mice treated with CCL21-expressing
but not control S. typhimurium. The anti-tumor activity was found to be dependent on CD4- and CD8-expressing cells, based on antibody-mediated in vivo immuno-depletion
experiments. Anti-tumor activity was achieved without evidence of toxicity. In summary, chemokine-expressing, attenuated bacteria
may provide a novel approach to cancer immunotherapy for effective and well-tolerated in vivo delivery of immunomodulatory
proteins.
Markus Loeffler and John C. Reed should be considered senior authors. 相似文献
12.
13.
A transgenic plant cell‐suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles
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Thangarasu Muthamilselvan Chin‐Wei Lee Yu‐Hsin Cho Feng‐Chao Wu Chung‐Chi Hu Yu‐Chuan Liang Na‐Sheng Lin Yau‐Heiu Hsu 《Plant biotechnology journal》2016,14(1):231-239
We describe a novel strategy to produce vaccine antigens using a plant cell‐suspension culture system in lieu of the conventional bacterial or animal cell‐culture systems. We generated transgenic cell‐suspension cultures from Nicotiana benthamiana leaves carrying wild‐type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot‐and‐mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co‐expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large‐scale production of immunopeptide vaccines in a cost‐effective manner using a plant cell‐suspension culture system. 相似文献
14.
As a step forward to achieve the generation of human complex-type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding
β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic
domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The
vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc1Man5GlcNAc2 and GlcNAc1Man3GlcNAc2, respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation
site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans. 相似文献
15.
Clementina Auriemma Maurizio Viscardi Simona Tafuri Luigi Michele Pavone Federico Capuano Laura Rinaldi Rossella Della Morte Giuseppe Iovane Norma Staiano 《Cellular & molecular biology letters》2010,15(3):496-506
Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host’s E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization
of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (ΔinlA) carrying a deletion in the gene coding for inlA. The ΔinlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-β3- and anti-β1-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-β2- and anti-β4-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial
uptake, showing different potencies correlated to their selective affinity for the β3- and β1-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes ΔinlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings
provide the first evidence that β3- and β1-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells. 相似文献
16.
S. Yu. Rakhmetova S. P. Radko O. V. Gnedenko N. V. Bodoev A. S. Ivanov A. I. Archakov 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2011,5(2):139-143
Aptamers interacting selectively with the anion-binding exosites 1 and 2 of thrombin were merged into dimeric oligonucleotide
constructs by means of a poly-(dT)-linker of 35 nucleotides (nt) in length. Complexes of thrombin with the aptamers and their
hetero- and homodimeric constructs were measured using an optical biosensor Biacore-3000. The K
D values obtained for the hetero- and homodimeric constructs were correspondingly 25–30- and 2–3-fold lower than those for
the primary aptamers. Analysis of temperature dependencies of the K
D values within the temperature interval of 10–40°C has shown that affinity increases with the temperature decrease. The values
of the enthalpy change ΔH upon formation of complexes of thrombin with the aptamers and the heterodimeric construct were basically the same. The value
of the entropy change ΔS upon complex formation of thrombin with the aptamer heterodimeric construct was 1.5–2-fold higher than the ΔS values for the complexes with the aptamers. The complex formation and dissociation rates increased with the elevation of
temperature from 10 to 37°C. However, at both temperatures the dissociation rate for the complex of thrombin with the heterodimeric
construct was evidently lower that for the complexes with the aptamers. 相似文献
17.
Seyed Javad Davarpanah Seo Hee Jung Yaw Joo Kim Youn-Il Park Sung Ran Min Jang Ryol Liu Won Joong Jeong 《Journal of Plant Biology》2009,52(3):244-250
Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species.
Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between
the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable
to investigations of the interactions between plastid and nucleus in N. benthamiana. 相似文献
18.
Jong Min Lee Ha Young Chung Kyung Il Kim Ki Hyun Yoo Jeon Hwang-Bo In Sik Chung Jong-Hwa Park 《Biotechnology letters》2011,33(1):41-46
We established a bicistronic expression system using an encephalomyocarditis virus (EMCV)-derived internal ribosomal entry
site (IRES) element to generate stably transformed Drosophila
melanogaster Schneider 2 (S2) cells expressing human rotavirus Wa capsid proteins, VP2 and VP6, for the synthesis of VP2/6 double-layered
virus-like particle (DVLP). The EMCV-derived IRES permitted bicistronic translation of recombinant VP6. Recombinant VP2 and
VP6 were detected in extracellular fractions of stably transformed S2 cells. A wheel-like DVLP (diam ~ 50–55 nm) with short
spikes was produced from the extracellular fraction of stably transformed S2 cells. A bicistronic expression system using
an EMCV-derived IRES element can thus be used to express two proteins of interest in stably transformed S2 cells. The bi-or
tri-cistronic expression of recombinant VP2/6/7 using stably transformed S2 cells can also be used to produce rotavirus VLPs. 相似文献
19.
A library of synthetic transcription activator‐like effector‐activated promoters for coordinated orthogonal gene expression in plants
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Kathleen Brückner Petra Schäfer Ernst Weber Ramona Grützner Sylvestre Marillonnet Alain Tissier 《The Plant journal : for cell and molecular biology》2015,82(4):707-716
20.
Alireza Shoae Hassani Feridon Malekzadeh Nour Amirmozafari Kasra Hamdi Negar Ordouzadeh Amir Ghaemi 《Current microbiology》2009,58(3):239-244
Exposure to ethanol is a stress condition that Salmonella typhimurium often encounters during its life cycle. Food, beverage, drugs, and cosmetics have a long history of using alcohols to control
pathogens. Ethanol is also commonly used for disinfecting medical instruments. This study was conducted to evaluate the ethanol
stress variations on the protein profile, cell structure, and serologic features of S. typhimurium. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed the phage shock protein G (pspG), a new ethanol-induced
stress protein in cells adapted to 10% ethanol. The result was confirmed by liquid chromatography–mass spectrometry. The maximum
quantity of this 9.02-kDa protein was produced in 12.5% (v/v) of ethanol-treated cultures. Scanning electron microscopy has
demonstrated new phenotypic characteristics in bacterial structure. The cells were unable to undergo binary fission. This
phenomenon explains the tight attachment of bacteria in a colony. Overall, ethanol extreme stress induced expression of new
proteins like PspG and repression of some other proteins in S. typhimurium. These induction and repression processes have inflicted dramatic changes on Salmonella behaviors.
Alireza Shoae Hassani, Kasra Hamdi and Amir Ghaemi are members of Young Researchers Club (YRC) of Tehran Science & Research
Campus of IAU, Tehran, Iran. 相似文献