title: A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.
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.
authors.affiliation: Clinical and Biotechnology B.U., Shimadzu Corporation, Kyoto, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Ezure
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Nanatani
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Suzuki
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Aizawa
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Souma
authors.affiliation: Clinical and Biotechnology B.U., Shimadzu Corporation, Kyoto, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Ito
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hohsaka
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
authors.forename: Gunnar
authors.initials: G
authors.lastname: von Heijine
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Utsumi
authors.affiliation: Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
authors.forename: Keietsu
authors.initials: K
authors.lastname: Abe
authors.affiliation: Clinical and Biotechnology B.U., Shimadzu Corporation, Kyoto, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Ando
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Uozumi
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007313:Insecta; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D000596:Amino Acids; D008565:Membrane Proteins