A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins
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{"title"=>"A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins", "type"=>"journal", "authors"=>[{"first_name"=>"Toru", "last_name"=>"Ezure", "scopus_author_id"=>"6603874907"}, {"first_name"=>"Kei", "last_name"=>"Nanatani", "scopus_author_id"=>"6504575846"}, {"first_name"=>"Yoko", "last_name"=>"Sato", "scopus_author_id"=>"55728704200"}, {"first_name"=>"Satomi", "last_name"=>"Suzuki", "scopus_author_id"=>"56438288200"}, {"first_name"=>"Keishi", "last_name"=>"Aizawa", "scopus_author_id"=>"55319422100"}, {"first_name"=>"Satoshi", "last_name"=>"Souma", "scopus_author_id"=>"56150987100"}, {"first_name"=>"Masaaki", "last_name"=>"Ito", "scopus_author_id"=>"57198911594"}, {"first_name"=>"Takahiro", "last_name"=>"Hohsaka", "scopus_author_id"=>"7004333801"}, {"first_name"=>"Gunnar", "last_name"=>"Von Heijine", "scopus_author_id"=>"56440223100"}, {"first_name"=>"Toshihiko", "last_name"=>"Utsumi", "scopus_author_id"=>"7101853524"}, {"first_name"=>"Keietsu", "last_name"=>"Abe", "scopus_author_id"=>"35314468800"}, {"first_name"=>"Eiji", "last_name"=>"Ando", "scopus_author_id"=>"7006444060"}, {"first_name"=>"Nobuyuki", "last_name"=>"Uozumi", "scopus_author_id"=>"7005703831"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"600703413", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0112874", "scopus"=>"2-s2.0-84915758120", "pmid"=>"25486605", "sgr"=>"84915758120"}, "id"=>"0748e6f1-d3c6-388a-b736-7091f0e31ade", "abstract"=>"Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.", "link"=>"http://www.mendeley.com/research/cellfree-translocation-system-using-extracts-cultured-insect-cells-yield-functional-membrane-protein", "reader_count"=>21, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Student > Doctoral Student"=>1, "Researcher"=>2, "Student > Ph. D. Student"=>5, "Student > Postgraduate"=>2, "Student > Master"=>3, "Student > Bachelor"=>5, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Student > Doctoral Student"=>1, "Researcher"=>2, "Student > Ph. D. Student"=>5, "Student > Postgraduate"=>2, "Student > Master"=>3, "Student > Bachelor"=>5, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>12, "Medicine and Dentistry"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>12}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>1}}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1830067"], "description"=>"<p>(A) Cleavage of the signal peptide of β-lactamase in microsomes. Translocation reactions containing <i>E. coli</i> β-lactamase labeled with FluoroTect Green<sub>Lys</sub> tRNA (12.5 µl) were mixed with 1 µl of sterilized water (lane 2), with 0.5 µl of 200 µg/ml proteinase K and 0.5 µl of 20% (v/v) Triton X-100 (lane 3) or 0.5 µl of 200 mg/ml proteinase K (lane 4). The samples were incubated for 1 h at 4°C. Equal volumes (6 µl) of the samples were separated by SDS-PAGE on 15% gels. The open circle marks the mature protein without the signal peptide. Lane 1 contains β-lactamase synthesized without microsomes as a control. (B) The N-terminal signal peptide sequence of β-lactamase is required for its translocation across the microsomal membrane. The <i>E. coli</i> β-lactamase lacking its signal peptide (Δsp-β-lactamase) and the wild type (β-lactamase) were labeled with FluoroTect Green<sub>Lys</sub> tRNA (12.5 µl). The translocation reaction was performed as in panel A. (C) N-linked glycosylation of membrane proteins in the microsomes. <i>Aeropyrum pernix</i> voltage-dependent K<sup>+</sup> channel (KvAP), human pro-tumor necrosis factor (pro-TNF) and human synaptobrevin II (Syb2) were labelled with <sup>35</sup>S-methionine during translation in the presence (+) or absence (-) of Sf21 microsomes. Dots indicate the glycosylated form of the proteins. (D) Confirmation of N-linked glycosylation of Syb2 labeled with <sup>35</sup>S-methionine. One µl of 10% (v/v) Triton X-100 and 1 µl of 500 mU/ml glycopeptidase F (GpF) were added to the translation mixture (12.5 µl), followed by incubation for 5 min at 37°C. The dot indicates the glycosylated protein.</p>", "links"=>[], "tags"=>["Sf 21 microsomes", "signal peptide cleavage", "Yield Functional Membrane Proteins", "polytopic membrane proteins", "Membrane proteins", "er", "insect cells", "Cultured Insect Cells", "translocation system"], "article_id"=>1264576, "categories"=>["Biological Sciences"], "users"=>["Toru Ezure", "Kei Nanatani", "Yoko Sato", "Satomi Suzuki", "Keishi Aizawa", "Satoshi Souma", "Masaaki Ito", "Takahiro Hohsaka", "Gunnar von Heijine", "Toshihiko Utsumi", "Keietsu Abe", "Eiji Ando", "Nobuyuki Uozumi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0112874.g002", "stats"=>{"downloads"=>3, "page_views"=>38, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Assessment_of_posttranslational_modification_of_secreted_proteins_and_membrane_proteins_synthesized_using_the_cell_free_system_containing_Sf21_microsomes_/1264576", "title"=>"Assessment of posttranslational modification of secreted proteins and membrane proteins synthesized using the cell-free system containing Sf21 microsomes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-08 03:10:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1830071"], "description"=>"<p>After synthesis of the membrane proteins in the cell-free system in the presence of FluoroTect Green<sub>Lys</sub> tRNA, the membrane fraction was precipitated and separated by SDS/PAGE. (A) 10% polyacrylamide gel; lane 1, AtKC1 (plant, K<sup>+</sup> channel, 6 transmembrane segments (TMS)); lane 2, KAT1 (plant, K<sup>+</sup> channel, 6 TMS); lane 3, KAT2 (plant, K<sup>+</sup> channel, 6 TMS); lane 4, GORK (plant, K<sup>+</sup> channel, 6 TMS); lane 5, AKT1 (plant, K<sup>+</sup> channel, 6 TMS); lane 6, NhaA (bacteria, Na<sup>+</sup>/H<sup>+</sup> antiporter, 10 TMS); Lane 7, AspT (bacteria, aspartate: alanine antiporter, 10 TMS); lane 8, MsbA (bacteria, ABC transporter, 12 TMS). (B) 15% polyacrylamide gel; lane 9, Sec61 β(human, 1 TMS).</p>", "links"=>[], "tags"=>["Sf 21 microsomes", "signal peptide cleavage", "Yield Functional Membrane Proteins", "polytopic membrane proteins", "Membrane proteins", "er", "insect cells", "Cultured Insect Cells", "translocation system"], "article_id"=>1264580, "categories"=>["Biological Sciences"], "users"=>["Toru Ezure", "Kei Nanatani", "Yoko Sato", "Satomi Suzuki", "Keishi Aizawa", "Satoshi Souma", "Masaaki Ito", "Takahiro Hohsaka", "Gunnar von Heijine", "Toshihiko Utsumi", "Keietsu Abe", "Eiji Ando", "Nobuyuki Uozumi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0112874.g003", "stats"=>{"downloads"=>2, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Production_of_different_membrane_proteins_using_the_cell_free_system_/1264580", "title"=>"Production of different membrane proteins using the cell-free system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-08 03:10:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1830074"], "description"=>"<p>(A) Cell-free synthesized AspT purified with cobalt affinity resin under non-denaturing conditions was solubilized with 1.5% DDM, and eluted with 0.01% DDM and 250 mM imidazole. The purified AspT protein (arrow head) was subjected to SDS-PAGE. (B) L-aspartate transport activity of proteoliposomes containing purified AspT protein. Proteoliposomes loaded with 100 mM L-aspartate were resuspended in 50 mM phosphate buffer (pH 7) (8.3 µg protein/ml). At 0 min, L-[<sup>3</sup>H] aspartate was added into the buffer (2.5 mM final concentration). After the rate of influx and efflux of L-[<sup>3</sup>H] aspartate was equal (at steady state), non-radiolabelled L-aspartate was added to a final concentration of 15 mM at 7.5 min (solid line, arrow indicating time of addition of unlabelled substrate). The broken line corresponds to the same experiment performed without addition of unlabelled L-aspartate. (C) Comparison of the initial uptake rates for L-aspartate into <i>E. coli</i> expressing AspT (hatched bar) and into microsomes containing cell-free synthesized AspT (solid bar). The transport activity at 1 min was regarded as the initial uptake rate.</p>", "links"=>[], "tags"=>["Sf 21 microsomes", "signal peptide cleavage", "Yield Functional Membrane Proteins", "polytopic membrane proteins", "Membrane proteins", "er", "insect cells", "Cultured Insect Cells", "translocation system"], "article_id"=>1264583, "categories"=>["Biological Sciences"], "users"=>["Toru Ezure", "Kei Nanatani", "Yoko Sato", "Satomi Suzuki", "Keishi Aizawa", "Satoshi Souma", "Masaaki Ito", "Takahiro Hohsaka", "Gunnar von Heijine", "Toshihiko Utsumi", "Keietsu Abe", "Eiji Ando", "Nobuyuki Uozumi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0112874.g004", "stats"=>{"downloads"=>2, "page_views"=>23, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Reconstitution_of_the_AspT_aspartate_transporter_synthesized_in_the_cell_free_system_with_Sf21_microsomes_/1264583", "title"=>"Reconstitution of the AspT aspartate transporter synthesized in the cell-free system with Sf21 microsomes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-08 03:10:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1830077"], "description"=>"<p>(A) Five version of NhaA (1–5) with introduced amber (TAG) codons (or Gly-Gly-TAG, introduced changes in bold) that enable incorporation of fluorescent non-natural amino acids into the N-terminal, middle or C-terminal regions of the protein (top panel) were expressed in the insect cell-free translation and translocation system in the presence of different fluorescent non-natural amino acids. The valine (V388) at the C-terminal end of NhaA was fused to additional sequences in the linker of the vectors (<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112874#pone-0112874-t001\" target=\"_blank\">Table 1</a>). Labeled NhaA protein was subjected to SDS-PAGE and fluorescent images were taken at the indicated excitation and emission wavelengths (bottom panel). The structure of the different fluorescent non-natural amino acids used in the experiments is shown above the gel images. (B) Incorporation of fluorescent non-natural amino acids into N-terminal or C-terminal positions in NhaA at an introduced four-base codon (CGGG) (top panel). NhaA was synthesized using the cell-free translation and translocation system in the presence of BODIPYFL-AF, subjected to SDS-PAGE and fluorescent images taken at the indicated excitation and emission wavelengths (bottom panel). (C) Double-labeling of NhaA by incorporation of BODIPY558-AF at an amber codon introduced into the N-terminal region and BODIPYFL-AF at the CGGG four-base codon introduced into the C-terminal region (top panel). NhaA was synthesized using the cell-free translation and translocation system in the presence of one or both fluorescent amino acids. Images of the SDS-PAGE were taken at the two different excitation and emission wavelengths indicated (bottom panel).</p>", "links"=>[], "tags"=>["Sf 21 microsomes", "signal peptide cleavage", "Yield Functional Membrane Proteins", "polytopic membrane proteins", "Membrane proteins", "er", "insect cells", "Cultured Insect Cells", "translocation system"], "article_id"=>1264586, "categories"=>["Biological Sciences"], "users"=>["Toru Ezure", "Kei Nanatani", "Yoko Sato", "Satomi Suzuki", "Keishi Aizawa", "Satoshi Souma", "Masaaki Ito", "Takahiro Hohsaka", "Gunnar von Heijine", "Toshihiko Utsumi", "Keietsu Abe", "Eiji Ando", "Nobuyuki Uozumi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0112874.g005", "stats"=>{"downloads"=>2, "page_views"=>27, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Incorporation_of_fluorescent_non_natural_amino_acids_into_NhaA_/1264586", "title"=>"Incorporation of fluorescent non-natural amino acids into NhaA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-08 03:10:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1830079"], "description"=>"<p>RNA templates encoding variants of KvAP, pro-TNF, or Syb2 were translated in the cell-free system supplemented with BODIPYFL-AF conjugated tRNA with or without the addition of Sf21microsomes (microsomes). The non-natural amino acids were incorporated into introduced TAG codon. The resulting proteins were subjected to SDS-PAGE (10-15% polyacrylamide) and detected with excitation at 488 nm and emission at 530 nm. The amber codon in the different constructs was substituted for the codon at the position corresponding to the listed amino acids (except for M6 in pro-TNF where the TAG codon was inserted as an additional codon after the M6 codon) and marked by a star in the diagram.</p>", "links"=>[], "tags"=>["Sf 21 microsomes", "signal peptide cleavage", "Yield Functional Membrane Proteins", "polytopic membrane proteins", "Membrane proteins", "er", "insect cells", "Cultured Insect Cells", "translocation system"], "article_id"=>1264588, "categories"=>["Biological Sciences"], "users"=>["Toru Ezure", "Kei Nanatani", "Yoko Sato", "Satomi Suzuki", "Keishi Aizawa", "Satoshi Souma", "Masaaki Ito", "Takahiro Hohsaka", "Gunnar von Heijine", "Toshihiko Utsumi", "Keietsu Abe", "Eiji Ando", "Nobuyuki Uozumi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0112874.g006", "stats"=>{"downloads"=>4, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Position_specific_incorporation_of_fluorescent_amino_acids_by_the_cell_free_system_/1264588", "title"=>"Position-specific incorporation of fluorescent amino acids by the cell-free system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-08 03:10:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1830081"], "description"=>"<p>List of constructs used.</p>", "links"=>[], "tags"=>["Sf 21 microsomes", "signal peptide cleavage", "Yield Functional Membrane Proteins", "polytopic membrane proteins", "Membrane proteins", "er", "insect cells", "Cultured Insect Cells", "translocation system"], "article_id"=>1264590, "categories"=>["Biological Sciences"], "users"=>["Toru Ezure", "Kei Nanatani", "Yoko Sato", "Satomi Suzuki", "Keishi Aizawa", "Satoshi Souma", "Masaaki Ito", "Takahiro Hohsaka", "Gunnar von Heijine", "Toshihiko Utsumi", "Keietsu Abe", "Eiji Ando", "Nobuyuki Uozumi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0112874.t001", "stats"=>{"downloads"=>3, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_List_of_constructs_used_/1264590", "title"=>"List of constructs used.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-08 03:10:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1830083"], "description"=>"<div><p>Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.</p></div>", "links"=>[], "tags"=>["Sf 21 microsomes", "signal peptide cleavage", "Yield Functional Membrane Proteins", "polytopic membrane proteins", "Membrane proteins", "er", "insect cells", "Cultured Insect Cells", "translocation system"], "article_id"=>1264592, "categories"=>["Biological Sciences"], "users"=>["Toru Ezure", "Kei Nanatani", "Yoko Sato", "Satomi Suzuki", "Keishi Aizawa", "Satoshi Souma", "Masaaki Ito", "Takahiro Hohsaka", "Gunnar von Heijine", "Toshihiko Utsumi", "Keietsu Abe", "Eiji Ando", "Nobuyuki Uozumi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0112874", "stats"=>{"downloads"=>7, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_Cell_Free_Translocation_System_Using_Extracts_of_Cultured_Insect_Cells_to_Yield_Functional_Membrane_Proteins_/1264592", "title"=>"A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-08 03:10:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/1830066"], "description"=>"<p>Fewer centrifugation and wash steps were required compared to the conventional protocol for the preparation of rough microsomes from dog pancreas and the composition of the buffer was simplified. Single-tube reactions consisting of Sf21microsomes, cell-free translationally active lysates from cultured insect cells and mRNA were performed to synthesize membrane proteins <i>in vitro</i>.</p>", "links"=>[], "tags"=>["Sf 21 microsomes", "signal peptide cleavage", "Yield Functional Membrane Proteins", "polytopic membrane proteins", "Membrane proteins", "er", "insect cells", "Cultured Insect Cells", "translocation system"], "article_id"=>1264575, "categories"=>["Biological Sciences"], "users"=>["Toru Ezure", "Kei Nanatani", "Yoko Sato", "Satomi Suzuki", "Keishi Aizawa", "Satoshi Souma", "Masaaki Ito", "Takahiro Hohsaka", "Gunnar von Heijine", "Toshihiko Utsumi", "Keietsu Abe", "Eiji Ando", "Nobuyuki Uozumi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0112874.g001", "stats"=>{"downloads"=>6, "page_views"=>375, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Flow_chart_for_the_preparation_of_microsomes_from_Sf21_cultured_insect_cells_/1264575", "title"=>"Flow chart for the preparation of microsomes from Sf21 cultured insect cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-08 03:10:39"}

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