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2013 Fiscal Year Research-status Report

New insights into protein folding based on bioinformatics analysis of cell-free protein synthesis

Research Project

Project/Area Number 25440023
Research Category

Grant-in-Aid for Scientific Research (C)

Research InstitutionKobe University

Principal Investigator

TOKMAKOV A・A  神戸大学, 学内共同利用施設等, 研究員 (20301278)

Project Period (FY) 2013-04-01 – 2016-03-31
Keywordsprotein folding / cell-free synthesis / bioinformatics
Research Abstract

Presently, an approach aimed at identification of numerous physicochemical, structural and functional properties of amino acid sequences, including the sites of multiple PTMs, associated with soluble expression of eukaryotic proteins in bacterial cell-free extracts has been finalized and published [1]. The developed method is intended for analysis of output from a cell-free protein production pipeline. It includes: 1) categorical assessment of expression data; 2) calculation and prediction of multiple properties of expressed sequences; 3) correlation of the individual properties with the expression scores; and 4) evaluation of statistical significance of the observed correlations.

Current Status of Research Progress
Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

At present, the method for identification of numerous physicochemical, structural and functional properties of amino acid sequences, including the sites of multiple PTMs, associated with soluble expression of eukaryotic proteins in bacterial cell-free extracts has been published [1]. The prototype of the prediction algorithm for assessment of protein amenability to cell-free expression is being evaluated by its ability to correctly classify new targets not present in the initial training set. The developed discriminant function is being applied to the test set of the expressed proteins, and prediction accuracy is estimated for this set.

Strategy for Future Research Activity

During the second year of study (2014) recombinant proteins will be expressed in the eukaryotic cell-free system. A complete dataset of cell-free expressed proteins will comprise a number of non-redundant (at 90% identity) amino acid sequences. The proteins of different functional and structural classes will be represented in the dataset, with the unbiased target selection in this regard. All the proteins will be expressed in the extracts under the same uniform set of conditions minimizing the influence of sequence-independent factors. The scores A, C, and N will be designated to all experimentally expressed proteins as follows: A - soluble proteins, C - expressed, but insoluble proteins, and N - non-expressed proteins. Multiple features of the expression sequences will be predicted using available bioinformatics tools.

  • Research Products

    (6 results)

All 2014 2013

All Journal Article (3 results) (of which Peer Reviewed: 3 results) Presentation (3 results)

  • [Journal Article] Bioinformatics analysis and optimization of cell-free protein synthesis2014

    • Author(s)
      2. Tokmakov AA, Kurotani A, Shirouzu M, Fukami Y, Yokoyama S.
    • Journal Title

      Methods Mol Biol

      Volume: v. 1118 Pages: 17-33

    • DOI

      10.1007/978-1-62703-782-2_2

    • Peer Reviewed
  • [Journal Article] Correlations between protein disorder and post-translational modifications in plants2014

    • Author(s)
      3. Kurotani A, Tokmakov AA, Kuroda Y, Fukami Y, Shinozaki K, Sakurai T.
    • Journal Title

      Bioinformatics

      Volume: v. 30 Pages: 1095-1103

    • DOI

      10.1093/bioinformatics/btt762

    • Peer Reviewed
  • [Journal Article] Monitoring gene expression in a single Xenopus oocyte using multiple cytoplasmic collections and quantitative RT-PCR2014

    • Author(s)
      1. Tokmakov AA, Hashimoto T, Hasegawa Y, Iguchi S, Iwasaki T, Fukami Y.
    • Journal Title

      FEBS Journal

      Volume: v. 281 Pages: 104-114

    • DOI

      10.1111/febs.12576

    • Peer Reviewed
  • [Presentation] Gene expression profiling in living Xenopus oocytes2013

    • Author(s)
      3. Alexander Tokmakov, Takanori Hashimoto, Sho Iguchi, Tetsushi Iwasaki, Yasuo Fukami
    • Organizer
      86th Annual Meeting of JBS
    • Place of Presentation
      Yokohama, Japan
    • Year and Date
      20130911-20130913
  • [Presentation] Gene expression profiling in single Xenopus oocytes and eggs2013

    • Author(s)
      2. Alexander A. Tokmakov, Takanori Hashimoto, Yushi Hasegawa, Sho Iguchi, Tetsushi Iwasaki and Yasuo Fukami
    • Organizer
      ISPROF 2013, 1st International Symposium on Profiling
    • Place of Presentation
      Costa da Caparica, Lisbon, Portugal
    • Year and Date
      20130902-20130904
  • [Presentation] mRNA degradation in apoptotic Xenopus eggs2013

    • Author(s)
      1. Alexander A. Tokmakov, Takanori Hashimoto, Yushi Hasegawa, Sho Iguchi, Tetsushi Iwasaki and Yasuo Fukami
    • Organizer
      Cold Spring Harbor symposium on Eukaryotic mRNA processing
    • Place of Presentation
      Cold Spring Harbor, New York, USA
    • Year and Date
      20130820-20130824

URL: 

Published: 2015-05-28  

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