2009 Fiscal Year Final Research Report
Characterization of cell-free protein synthesis system and theapplication for the synthesis of cell membrane protein
| Project/Area Number |
17076011
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | National Institute of Advanced Industrial Science and Technology (2006-2009)
The University of Tokushima (2005) |
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Principal Investigator |
KATAOKA Masaroshi National Institute of Advanced Industrial Science and Technology, 健康工学研究センター, 研究チーム長 (20224438)
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| Co-Investigator(Kenkyū-buntansha) |
SHINOHARA Yasuo 徳島大学, 疾患ゲノム研究センター, 教授 (60226157)
ISHIDA Tatsuhiro 徳島大学, 大学院・ヘルスバイオサイエンス研究部, 准教授 (50325271)
MORI Shuji 岡山大学, 医歯薬総合研究科, 准教授 (50220009)
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| Project Period (FY) |
2005 – 2009
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| Keywords | 膜タンパク質 / リポソーム / 試験管内タンパク質合成 / コートペプチド / 細胞機能模倣 / 再構成 |
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| Research Abstract |
We reconstructed the membrane protein for the mimicking the cell function. During the replication processes, coat proteins of Pf3 phage is synthesized in the bacteria and inserted into bacteria membrane. Since one mutant of coat protein Pf3 phages, 3L-Pf3, having longer hydrophobic region could be inserted into lipid bilayer membrane even in the absence of membrane proteins, studies on the interaction of coat protein of bacterial phages with lipid bilayer membrane are expected to fundamental principles on the formation of membrane protein. Using 3L-Pf3 as a template, we prepared two mutants of 3L-DR and 3L-RD. Modification of the peptide with His-tag enabled the simple and rapid purification of the synthesized peptide, and those with T7and Myc-tags enabled clear determination of their topologies in the liposomal membrane. The two peptides were found to be spontaneously and directionally inserted into negatively charged large unilamellar vesicle, according to the charge distribution in the peptides. Substitution of amino acids in a peptide caused remarkable differences in their immunoreactivities with antidodies. Observed differences in immunoreactivities among the peptides were not due to the differences in efficiencies of their transfer onto nitrocellulose or PVDF membranes. Rather, possible folding of the peptide on the membrane was considered to be the reason for their distinct immunoreactivities with the antibodies.
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[Journal Article] Ca2+-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions2009
Author(s)
Yamada A, Yamamoto T, Yoshimura Y, Gouda S, Kawashima S, Yamazaki N, Yamashita K, Kataoka M, Nagata T, Terada H, Pfeiffer DR, Shinohara Y
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Journal Title
Biochim Biophys Acta 1787(12)
Pages: 1486-91
Peer Reviewed
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[Journal Article] Accurate quantitation of salivary and pancreatic amylase activities in human plasama by microchip electrophoretic separation of the substrates and hydrolysates coupled with immunoinhibition2008
Author(s)
Maeda E, Kataoka M, Yatsushiro S, Kajimoto K, Hino M, Kaji N, Tokeshi M, Bando M, Kido J, Ishikawa M, Shinohara Y, Baba Y.
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Journal Title
Electrophoresis 29(9)
Pages: 1902-9
Peer Reviewed
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[Journal Article] Functionally important conserved length of C-terminal regions of yeast and bovine ADP/ATP carriers, identified by deletion mutants studies, and water accessibility of the amino acids at the C-terminal region of the yeast carrier2008
Author(s)
Iwahashi A, Ishii A, Yamazaki N, Hashimoto M, Ohkura K, Kataoka M, Majima E, Terada H, Shinohara Y.
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Journal Title
Mitochondrion 8(2)
Pages: 196-204
Peer Reviewed
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[Journal Article] Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis2007
Author(s)
Kakuhata R, Watanabe M, Yamamoto T, Akamine R, Yamazaki N, Kataoka M, Fukuoka S, Ishikawa M, Ooie T, Baba Y, Hori T, Shinohara Y.
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Journal Title
J Biochem Biophys Methods 70(5)
Pages: 755-60
Peer Reviewed
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[Journal Article] Usefulness of the 5' region of the cDNA encoding acidic ribosomal phosphoprotein P0 conserved among rats, mice, and humans as a standard probe for gene expression analysis in different tissues and animal species2007
Author(s)
Akamine R, Yamamoto T, Watanabe M, Yamazaki N, Kataoka M, Ishikawa M, Ooie T, Baba Y, Shinohara Y.
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Journal Title
J Biochem Biophys Methods 70(3)
Pages: 481-6
Peer Reviewed
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[Journal Article] VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues2006
Author(s)
Yamamoto T, Yamada A, Watanabe M, Yoshimura Y, Yamazaki N, Yoshimura Y, Yamauchi T, Kataoka M, Nagata T, Terada H, Shinohara Y
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Journal Title
J Proteome Res 5(12)
Pages: 3336-44
Peer Reviewed
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