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RBD JAV Yui Hatano, Yuka Minase, dan Nao Kojima dalam Wanita sudah Menikah Perawat Enggan Cuplikan Gratis dan Subtitle Bahasa Indonesia Ang isang insidente ay nangyayari sa isang pangkalahatang ospital sa sikat ng araw. Isang lalaki na isang pasyente sa ospital na pessimistic sa kanyang kondisyong medikal at naging desperado, nang-hostage sa mga nars at pasyente na naroroon sa ward, at ang asawa ng pasyente na bumibisita sa kanya. Ano ang kapalaran ng asawa ng isang inpatient na HIGH SPEED DOWNLOAD ; Download type: Free: Premium: Download speed
SIMG 2 Nov WSP 21 Jun This is an open-access article distributed under the terms of the Creative Commons Attribution License CC BY.The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.No use, distribution or reproduction is permitted which does not comply with these terms.strasser boku.Plant Glycobiology A Sweet World of Glycans, Glycoproteins, Glycolipids, and Carbohydrate-Binding Proteins.Open supplemental data Export citation EndNote Reference Manager Simple TEXT file BibTex.Check for updates.ORIGINAL RESEARCH article.Introduction The ongoing COVID pandemic underscores the urgency to increase the preparedness for future virus outbreaks and to establish countermeasures such as platform technologies to produce recombinant proteins for subunit vaccines or viral antigens for diagnostic tests Amanat et al.Results RBD Is Present as a Homodimer and Non-functional After Purification From N.benthamiana The receptor-binding domain RBD of the SARS-CoV-2 spike protein amino acids RF Figure 1A and Supplementary Figure 1 Amanat et al.x PubMed Abstract CrossRef Full Text Google Scholar.M PubMed Abstract CrossRef Full Text Google Scholar.abb PubMed Abstract CrossRef Full Text Google Scholar.Edited by: Nobuyuki Matoba , University of Louisville, United States.This article is part of the Research Topic Plant Glycobiology A Sweet World of Glycans, Glycoproteins, Glycolipids, and Carbohydrate-Binding Proteins View all 33 Articles.People also looked at. srt Translation: Terjemahan Manusia bukan A.Resolusi Video dan Ukuran File p HD 4, MB p HD 3, MB p 2, MB p 1, MB p MB p MB.Pertanyaan yang Sering Diajukan Bagaimana cara mengunduh video lengkapnya? BF 2 Nov Saberianfar, R.Protein body formation in leaves of Nicotiana benthamiana : a concentration-dependent mechanism influenced by the presence of fusion tags.Saijo, Y.Receptor quality control in the endoplasmic reticulum for plant innate immunity.EMBO J.Sainsbury, F.pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants.Schillberg, S.Critical analysis of the commercial potential of plants for the production of recombinant proteins.Schoberer, J.The golgi localization of GnTI requires a polar amino acid residue within its transmembrane domain.Plant Physiol.Shajahan, A.Deducing the N- and O- glycosylation profile of the spike protein of novel coronavirus SARS-CoV Anda hanya perlu melakukan "Pesanan Subtitel Khusus" untuk subtitel dan kami akan membuatnya dan mengirimkannya dalam 5 9 hari.Untuk memesan subtitle RBD, klik tombol 'Pesan' di bagian atas halaman ini.Bagaimana Anda mengenakan biaya untuk pesanan subtitel khusus? Here, we investigated the role of N-glycosylation for expression and function of the receptor binding domain RBD from the SARS-CoV-2 spike protein.Recombinant RBD can be used for vaccination approaches Yang et al.The SARS-CoV-2 RBD has two N-glycosylation sites N and N that are fully glycosylated when expressed in heterologous expression systems Antonopoulos et al.Our data show that N-glycans on the RBD from the SARS-CoV-2 spike protein are important for protein folding and efficient RBD production as functional protein.The receptor-binding domain RBD of the SARS-CoV-2 spike protein amino acids RF Figure 1A and Supplementary Figure 1 Amanat et al.A band of approximately 34 kDa was detectable and the expression levels were comparable in both N.benthamiana lines Figure 1B.During purification, we noticed that the protein was less stable and prone to aggregate.Purified plant-derived RBD migrated faster on immunoblots compared to RBD produced in HEK cells RBD-HEK which is presumably caused by divergent N-glycan processing Figure 1D.SDS-PAGE under non-reducing conditions followed by immunoblotting revealed a major band of approximately 50 kDa for plant-produced RBD, indicating the presence of dimers.Homodimer formation for plant and mammalian-cell produced RBD was recently confirmed by size-exclusion chromatography and is likely promoted by incorrectly formed disulfide bonds Klausberger et al.Figure 1.SARS-CoV-2 RBD is poorly expressed in Nicotiana benthamiana.A Schematic illustration of the SARS-CoV-2 RBD variant that was expressed.The position of the signal peptide SP , the two N-glycans at position N and N and the C-terminal 6x histidine tag are indicated.B Protein extracts from infiltrated N.D Immunoblot analysis of RBD produced in HEK cells RBD-HEK, — aa and RBD produced in N.SDS-PAGE was carried out under reducing and non-reducing conditions.Binding was analyzed using a Luminex bead-based assay and the median fluorescent intensity log2 MFI is shown.To examine whether plant-produced RBD is correctly folded, we analyzed the binding to human angiotensin converting enzyme 2 ACE2 by ELISA.While RBD-HEK displayed the expected interaction with an ACE2-Fc fusion protein see below , binding by plant-produced RBD was not consistently observed data not shown.Similar results were obtained when the monoclonal antibody CR was used as a capture antibody.CR binds to a conformational RBD epitope Yuan et al.Despite the failure to bind ACE2-Fc and CR, plant-produced RBD reacted with convalescent sera indicating the presence of epitopes that are recognized by polyclonal antibodies present in SARS-CoV-2 exposed individuals Figure 1E.To see if increased ER-retention is beneficial for RBD production, we expressed RBD-KDEL transiently in N.benthamiana leaves Figure 2A.Surprisingly, RBD-KDEL, which differs from RBD only by the presence of the KDEL tetrapeptide inserted after the polyhistidine tag Supplementary Figure 1 , was not detectable in crude protein extracts from infiltrated leaves Figure 2B , lane 1.When we co-expressed human CRT, on the other hand, we could clearly detect RBD-KDEL on immunoblots Figures 2B,D.By contrast, co-expression of Arabidopsis CRT2, which stabilized the ERAD substrate SUBEX-C57Y-GFP Figure 2E , or co-expression of Arabidopsis CNX1, did not have an impact on RBD-KDEL Figure 2D and Supplementary Figure 2.Taken together, these data indicate that RBD-KDEL is poorly expressed as a soluble protein in N.Figure 2.Co-expression of human CRT results in RBD-KDEL accumulation in Nicotiana benthamiana.A Schematic illustration of the expressed SARS-CoV-2 RBD-KDEL variant.Samples were analyzed by immunoblotting 3 days after infiltration of N.benthamiana WT.Ponceau S staining Ponc.is shown as a loading control.RBD expression was included for comparison.C SUBEX-C57Y-GFP was co-expressed with kif or Arabidopsis CDC48A-QQ QQ and analyzed 3 days after infiltration of N.D RBD-KDEL was co-expressed with HsCRT or Arabidopsis CRT2 AtCRT2.Both CRT variants were expressed with the pEAQ- HT vector and expression was analyzed 4 days after infiltration of N.E SUBEX-C57Y-GFP was co-expressed with HsCRT or AtCRT2 and analyzed 3 days after infiltration of N.Next, we examined the effect of human CRT on RBD expression.When we co-expressed human CRT, the RBD expression levels appeared unchanged, but the mobility in SDS-PAGE was altered indicating differences in N-glycan processing Figure 3A.We hypothesized that binding of human CRT prevents the trimming of monoglucosylated N-glycans Glc 1 Man 9 GlcNAc 2 to complex ones and causes the observed slower migration.Upon Endo H digestion a shift in mobility was detectable showing that RBD co-expressed with human CRT harbors oligomannosidic N-glycans Figure 3B.The additional RBD bands that are detectable with the anti-RBD antibody are likely the result of proteolytic processing at the C-terminus Figure 3A which might take place in the apoplast.Figure 3.Human CRT retains glycoproteins in intracellular compartments.A HsCRT was co-expressed with RBD and analyzed 4 days after infiltration of Nicotiana benthamiana WT by immunoblotting with antibodies against RBD or the 6x histidine tag.B Endo H digestion of RBD co-expressed with.C Confocal microscopy of RBD-RFP co-expressed with HsCRT, AtCNX1 or AtCRT2.D Enlarged confocal image of RBD-RFP co-expressed with HsCRT.E Endo H digestion and immunoblotting of RBD-RFP co-expressed with HsCRT.Images were taken 3 days after infiltration.The previous experiments indicated that RBD is trafficking through the Golgi where oligomannosidic N-glycans are processed to complex ones Figure 1B.Co-expression of human CRT prevents the maturation to complex N-glycans by retaining RBD in the ER or by protecting the N-glycans from processing in the Golgi.To examine the effect of CRT on the subcellular localization of RBD we replaced the polyhistidine-tag on RBD with the red fluorescent protein RFP and analyzed its localization in N.benthamiana leaf epidermal cells.Confocal microscopy confirmed that RBD-RFP was secreted to the apoplast Figure 3C.By contrast, in the presence of human CRT, RBD-RFP was found in intracellular structures visually resembling protein bodies Conley et al.RBD-RFP expressed with human CRT displayed Endo H sensitive N-glycans, suggesting that the intracellular structures are ER-derived Figure 3E.Arabidopsis CRT2 or CNX1 expression did not cause the localization in such cellular structures and RBD-RFP was still secreted to the apoplast Figure 3C.To test if human CRT is specific for RBD or generally retaining glycoproteins in protein body-like structures, we co-expressed two glycoproteins, Arabidopsis RFP-PDI5 and N.benthamiana HEXO3-RFP Farid et al.While the two glycoproteins were found in protein body-like structures, ST-RFP was still detected in the apoplast Figure 3F.This shows that human CRT specifically retains glycoproteins in ER-derived intracellular protein body-like structures but does not have a general effect on secretion of proteins.The low expression levels of RBD and the tendency for homodimer formation is possibly caused by the presence of an unpaired cysteine residue C at the C-terminus Supplementary Figure 1 leading to intermolecular cross-linking, homodimer formation and aggregation.To improve the expression of the soluble monomeric form we expressed an RBD variant with the cysteine at position substituted by an alanine residue transiently in N.While RBD-CA could be detected on immunoblots with an RBD-specific polyclonal antibody, reduced signals were present on immunoblots probed with an anti-His antibody indicating that the C-terminus is either not accessible or unstable Supplementary Figure 3.Moreover, SDS-PAGE separation under non-reducing conditions and immunoblot analysis with an RBD-specific antibody showed that considerable amounts of dimeric RBD-CA are still present, despite the removal of the unpaired cysteine residue.This result indicates that other cysteines may also contribute to the formation of dimeric RBD variants.Next, we expressed a truncated RBD variant RBD amino acids RL lacking the cysteine at position Figure 4A and Supplementary Figure 1.MS analysis of peptides showed that both RBD N-glycosylation sites contained exclusively Golgi-processed N-glycans Supplementary Figure 5.GlcNAc 2 Man 3 GlcNAc 2 GnGn was the major peak on both sites and low amounts of truncated N-glycans likely generated in the apoplast were present.A bead-based binding assay with sera from SARS-CoV-2 exposed individuals confirmed that recombinant RBD is recognized as SARS-CoV-2 antigen with high sensitivity and specificity Figure 4D.ELISA with ACE2-Fc and the conformation-dependent RBD antibody CR showed that RBD binds equally well like HEKproduced RBD to ACE2-Fc and CR Figures 4E,F.Figure 4.A plant-produced truncated RBD variant is functional.A Schematic illustration of the truncated RBD variant.C RBD variants were purified from the apoplastic fluid 4 days after infiltration, analyzed by SDS-PAGE under reducing or non-reducing conditions, followed by Coomassie Brilliant Blue CBB staining.HEKproduced RBD RBD-HEK was included for comparison.The altered mobility of RBD-HEK and the plant produced RBD variant is caused by differences in complex N-glycans.The arrow marks the position of the homodimer.E Binding of purified plant-produced RBD and RBD-HEK to plates coated with ACE2-Fc or F antibody CR