Recombinant Production of the Amino Terminal Cytoplasmic Region of Dengue Virus Non-Structural Protein 4A for Structural Studies
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{"title"=>"Recombinant production of the amino terminal cytoplasmic region of dengue virus non-structural protein 4A for structural studies", "type"=>"journal", "authors"=>[{"first_name"=>"Yu Fu", "last_name"=>"Hung", "scopus_author_id"=>"55914189100"}, {"first_name"=>"Olga", "last_name"=>"Valdau", "scopus_author_id"=>"55633810400"}, {"first_name"=>"Sven", "last_name"=>"Schünke", "scopus_author_id"=>"26537977600"}, {"first_name"=>"Omer", "last_name"=>"Stern", "scopus_author_id"=>"8866206600"}, {"first_name"=>"Bernd W.", "last_name"=>"Koenig", "scopus_author_id"=>"7005202358"}, {"first_name"=>"Dieter", "last_name"=>"Willbold", "scopus_author_id"=>"7004563027"}, {"first_name"=>"Silke", "last_name"=>"Hoffmann", "scopus_author_id"=>"7202629745"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"373022565", "pmid"=>"24466115", "doi"=>"10.1371/journal.pone.0086482", "scopus"=>"2-s2.0-84899813573", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "issn"=>"19326203", "sgr"=>"84899813573"}, "id"=>"00ad0591-fc99-30f6-8406-a57faf568e25", "abstract"=>"BACKGROUND: 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 amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. Its amphipathic character was shown to be essential for viral replication. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: This report describes the optimization of a uniform procedure for the expression and purification of the wild type NS4A(1-48) peptide and a peptide derived from a replication-deficient mutant NS4A(1-48; L6E, M10E) with disrupted amphipathic nature. A codon-optimized, synthetic gene for NS4A(1-48) was expressed as a fusion with a GST-GB1 dual tag in E. coli. Tobacco etch virus (TEV) protease mediated cleavage generated NS4A(1-48) peptides without any artificial overhang. Using the described protocol up to 4 milligrams of the wild type or up to 5 milligrams of the mutant peptide were obtained from a one-liter culture. Isotopic labeling of the peptides was achieved and initial NMR spectra were recorded.\\n\\nCONCLUSIONS/SIGNIFICANCE: Small molecules targeting amphipathic helices in the related Hepatitis C virus were shown to inhibit viral replication, representing a new class of antiviral drugs. These findings highlight the need for an efficient procedure that provides large quantities of the amphipathic helix containing NS4A peptides. The double tag strategy presented in this manuscript answers these needs yielding amounts that are sufficient for comprehensive biophysical and structural studies, which might reveal new drug targets.", "link"=>"http://www.mendeley.com/research/recombinant-production-amino-terminal-cytoplasmic-region-dengue-virus-nonstructural-protein-4a-struc", "reader_count"=>20, "reader_count_by_academic_status"=>{"Researcher"=>6, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>3, "Student > Master"=>3, "Student > Bachelor"=>5, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Researcher"=>6, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>3, "Student > Master"=>3, "Student > Bachelor"=>5, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>10, "Medicine and Dentistry"=>7, "Chemistry"=>1, "Social Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>7}, "Chemistry"=>{"Chemistry"=>1}, "Social Sciences"=>{"Social Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>10}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}}, "reader_count_by_country"=>{"South Africa"=>1, "Mexico"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1357669"], "description"=>"<p>(A) Relative expression levels of GST-GB1-NS4A(1–48) and the GST-GB1-NS4A(1–48; L6E, M10E) mutant were analyzed by SDS-PAGE using aliquots of the expression cultures. Shown are samples obtained from culture at 0 (0) or 3 hours (I) following IPTG induction or the supernatant after cell lysis (S). (B) TEV digest of GST-GB1-NS4A(1–48) wild type and mutant protein fusions. Aliquots of GSH-purified supernatants of wild type and mutant fusion proteins before and after TEV cleavage are shown. Note that besides the GST-GB1-NS4A(1–48) full-length product also shorter fragments, likely GST-GB1 and other truncation fragments, marked by asterisks were produced, which are present in the GSH-purified samples already prior to TEV cleavage. Because staining of free NS4A(1–48) peptides is very faint under the conditions used, the progress of the TEV digest is monitored by observing the decrease of the band for the dual tagged GST-GB1-NS4A(1-48) fusion protein in parallel with an increase of the band for the free GST-GB1 dual tag. A densitometric analysis of the respective bands revealed a cleavage efficiency of approximately 50% for the wild type peptide.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue", "mutant", "peptides", "dual", "gst-gb1"], "article_id"=>909965, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.g004", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_NS4A_1_8211_48_wild_type_and_mutant_peptides_using_a_dual_GST_GB1_tag_/909965", "title"=>"Expression of NS4A(1–48) wild type and mutant peptides using a dual GST-GB1 tag.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-23 03:12:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1357668"], "description"=>"<p>The GB1-NS4A(1–48) sequence was inserted into pGEX-4T-2 between the <i>Eco</i>RI and <i>Xho</i>I sites. Protease recognition motifs are underlined while the cleavage sites are marked by triangles. The two thrombin sites, which originate from the vector backbone of pGEX and pGEV, respectively, are shown in brackets and were not used in our protocol. Note that TEV digestion produces a native NS4A peptide.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue"], "article_id"=>909964, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.g003", "stats"=>{"downloads"=>2, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_cloning_region_of_pGEX_GB1_NS4A_1_8211_48_/909964", "title"=>"Expression/cloning region of pGEX-GB1-NS4A(1–48).", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-23 03:12:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1357673"], "description"=>"<p>Note: The sequences of the restriction sites used for cloning are underlined, the name of the enzyme is given below.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue", "amplification", "ns4a"], "article_id"=>909969, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.t001", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primers_used_for_amplification_of_NS4A_1_8211_48_/909969", "title"=>"Primers used for amplification of NS4A (1–48).", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-01-23 03:12:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1357671"], "description"=>"<p>The 15% SDS-PAGE gel containing samples from various steps in the purification procedure is shown on the left. The wild type peptide is shown in (A) while the mutant is shown in (B). The supernatant after cell lysis is shown in lane 1. The lysate containing the peptide was loaded on a GSH sepharose column (lane 2), and cleaved by TEV protease on the column, the cleaved protein is shown in lane 3. The GSTfusion tag remained bound to the column, while the NS4A(1–48) peptide was collected from the flow-through (lane 4). The peptide was further purified by size exclusion chromatography (lane 5). The mutant peptide was purified using the same strategy (B). The respective size exclusion chromatography profiles (HiLoad 16/60 Superdex 75 prep grade) of the flow-through fraction from the TEV protease on column cleavage step - mainly containing TEV protease and NS4A(1–48; L6E, M10E) or NS4A(1–48) peptides - are shown on the right with the matching SDS-PAGE analysis of the NS4A containing fractions.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue", "recombinant", "mutant"], "article_id"=>909967, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.g006", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Purification_of_recombinant_NS4A_1_8211_48_wild_type_and_mutant_peptides_/909967", "title"=>"Purification of recombinant NS4A(1–48) wild type and mutant peptides.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-23 03:12:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1357672"], "description"=>"<p>Spectrum of 0.5[<sup>15</sup>N]-NS4A(1–48) wild type (A) and of 1 mM mutant (L6E, M10E) peptide (B) in 50 mM sodium phosphate buffer, pH 6.8. Data were recorded at 30°C.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue", "2d", "spectra", "purified", "ns4a"], "article_id"=>909968, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.g007", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Recorded_2D_1_H_15_N_BEST_TROSY_spectra_of_the_purified_NS4A_peptides_/909968", "title"=>"Recorded 2D (<sup>1</sup>H, <sup>15</sup>N)-BEST-TROSY spectra of the purified NS4A peptides.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-23 03:12:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1357670"], "description"=>"<p>(A) A schematic model explaining the resistance of the GST-GB1 dual tagged wild type NS4A(1–48) peptide to TEV cleavage. Scissors illustrate the protease while the triangles represent the position of the cleavage sites. GST induced oligomerization of wild type NS4A(1 48) may block the protease cleavage site. (B) Analysis of the urea tolerance of TEV protease activity. GST-GB1 tag removal from NS4A(1 48; L6E, M10E) in the presence of different concentrations of urea (M). Fusion peptides were incubated with TEV protease at 20°C for 16 h at a fusion peptide to protease molar ratio of approximately 100. (C) Binding properties of GST-GB1-NS4A(1–48) to GSH sepharose in the presence of urea. Equal amounts of the fusion peptide were subjected to a mini-scale GSH-affinity chromatography in the presence of different concentrations of urea (M). Input, flow-through and elution fractions were analyzed by 15% SDS-PAGE. (D) TEV cleavage efficiency of wild type NS4A(1–48) peptide in the presence of urea. GST-GB1 tag removal from NS4A(1–48) in the presence of different concentrations of urea given in Molars. Fusion peptides were incubated in the absence (−) or presence (+) of TEV protease at 20°C for 16 h. The different “+” font sizes indicate the increasing amounts of TEV protease with fusion peptide to protease molar ratios of approximately 100, 50 and 10. The progress of the TEV digest is monitored by observing the decrease of the band of the dual tagged GST-GB1-NS4A(1–48) fusion protein and a parallel increase of the free GST-GB1 dual tag band (B, D).</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue", "tev", "protease", "cleavage"], "article_id"=>909966, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.g005", "stats"=>{"downloads"=>11, "page_views"=>300, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Optimization_of_TEV_protease_cleavage_conditions_/909966", "title"=>"Optimization of TEV protease cleavage conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-23 03:12:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1357667"], "description"=>"<p>(A) SDS-PAGE analysis of the relative expression levels of NS4A fusions with ubiquitin (Ubi-NS4A(1–48)), glutathion-S-transferase (GST-NS4A(1–48)) and immunoglobulin-binding domain of streptococcal protein G (GB1-NS4A(1–48)). Aliquots of the expression cultures taken before (0) or 3 hours after IPTG induction (I) were applied. Aliquots of the supernatants after cell lysis (S) are shown as well. (B) TEV cleavage of the purified GB1-NS4A(1–48) fusion protein. Purified GB1-NS4A(1–48) fusion protein after size exclusion chromatography before (−) and after (+) TEV digestion together with a molecular weight marker (M; M3546, Sigma) were applied.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue", "constructs", "containing", "fusion"], "article_id"=>909963, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.g002", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparative_expression_of_NS4A_1_8211_48_constructs_containing_single_fusion_tags_/909963", "title"=>"Comparative expression of NS4A(1–48) constructs containing single fusion tags.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-23 03:12:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1357666"], "description"=>"<p>ClustalW alignment <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086482#pone.0086482-Larkin1\" target=\"_blank\">[40]</a> of the NS4A(1–48) coding sequence as found in the viral genome of DENV type 2 and following optimization for <i>E. coli</i> expression <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086482#pone.0086482-Fath1\" target=\"_blank\">[21]</a>.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue", "coding"], "article_id"=>909962, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.g001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_NS4A_1_8211_48_coding_sequence_/909962", "title"=>"NS4A(1–48) coding sequence.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-23 03:12:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1357674"], "description"=>"<p>Due to the different sizes of the fusion tags (Ubi: 11.3 kDa, GST: 26.4 kDa, GB1: 7.6 kDa, GST-GB1: 34 kDa) the mass fractions of the target peptide differ significantly between the constructs (Ubi: 31.5%, GST: 16%, GB1: 40.6%, GST-GB1: 13.3%). Since Ubi and GST fusion constructs resulted in very low yields (>1 mg/l), data for these constructs have not been included. The values for GB1 and GST-GB1 have been used to calculate the theoretical NS4A(1–48) content after the first purification step for each of the constructs shown. Despite an unfavorable target peptide to fusion tag ratio highest recovery and purity values were obtained with the dual GST-GB1-NS4A(1–48) construct after proteolytic cleavage and tag removal. LB medium was used if not stated otherwise. An asterisk (*) indicates isotope ([<sup>13</sup>C, <sup>15</sup>N] or [<sup>15</sup>N]) labeled minimal growth medium. Note that the overall peptide yields were higher in minimal medium compared to those in rich LB medium. Amount and purity of the fusion protein tagged peptides as well as the purity of the free peptides has been estimated from SDS PAGE analysis. Final free peptide yields were additionally calculated by measuring the concentration of the peptides at 280 nm using an extinction coefficient of 1490 M<sup>−1</sup> cm<sup>−1</sup> in water.</p>", "links"=>[], "tags"=>["Biochemistry", "proteins", "protein structure", "Recombinant proteins", "Transmembrane proteins", "biotechnology", "drug discovery", "microbiology", "Virology", "Viral classification", "RNA viruses", "proteomics", "Spectrometric identification of proteins", "Infectious diseases", "Viral diseases", "dengue", "yields", "purities", "studied", "fusion", "peptides"], "article_id"=>909970, "categories"=>["Biological Sciences", "Medicine"], "users"=>["Yu-Fu Hung", "Olga Valdau", "Sven Schünke", "Omer Stern", "Bernd W. Koenig", "Dieter Willbold", "Silke Hoffmann"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0086482.t002", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Average_yields_and_purities_of_the_studied_NS4A_1_8211_48_fusion_proteins_and_of_the_resulting_NS4A_1_8211_48_target_peptides_obtained_from_1_L_of_culture_/909970", "title"=>"Average yields and purities of the studied NS4A(1–48) fusion proteins and of the resulting NS4A(1–48) target peptides obtained from 1 L of culture.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-01-23 03:12:59"}

PMC Usage Stats | Further Information

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