2014 Fiscal Year Research-status Report
New insights into protein folding based on bioinformatics analysis of cell-free protein synthesis
| Project/Area Number |
25440023
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| Research Institution | Kobe University |
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Principal Investigator |
TOKMAKOV A・A 神戸大学, 遺伝子実験センター, 客員教授 (20301278)
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| Project Period (FY) |
2013-04-01 – 2016-03-31
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| Keywords | protein folding / cell-free synthesis / bioinformatics |
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| Outline of Annual Research Achievements |
A method for identification of multiple physicochemical and structural properties associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts has been published [1]. The method 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. Using this approach, a number of significant correlations between calculated and predicted properties of amino acid sequences and their propensity for soluble cell-free expression have been revealed. The results of these studies were presented at the EMBO International Conference and ICPAC 2014 International Meeting [1, 2]. Also, the expression of eukaryotic proteins in a eukaryotic (insect) cell-free system has been studied and a manuscript was submitted to an international journal.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The focus is set on the analysis of cell-free expression of eukaryotic proteins in an insect cell-free system. Significant differences between prokaryotic and eukaryotic cell-free expression have been revealed. They are associated primarily with the lack of certain post-translational modifications in bacterial extracts, as well as with different reducing conditions in the cytoplasm of eukaryotic and prokaryotic cells. The presence of intrinsically disordered motifs in the amino acid sequences was found to greatly affect the success rate of cell-free protein synthesis both in eukaryotic and prokaryotic cell-free expression systems.
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| Strategy for Future Research Activity |
At the third year of study, the analysis of eukaryotic cell-free protein synthesis will be be continued. Identification of protein properties that correlate best with successful expression in a eukaryotic system of cell-free protein synthesis will be done for the purpose of development of an algorithm for predicting protein amenability to cell-free eukaryotic expression. The comparison of prokaryotic and eukaryotic cell-free protein synthesis will be done. Special attention will be paid to the effects of post-translational modifications on the expression efficiency and protein folding. Also, the presence of intrinsically disordered motifs in the amino acid sequences will be correlated to the success rate (expression yield and solubility) of cell-free protein synthesis both in eukaryotic and prokaryotic systems.
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| Causes of Carryover |
The money leftover from the previous fiscal year (111,481 yen) will be used in the next fiscal year. It is necessary to boost a relatively small budget of 1,200,000 yen for the final year of this study.
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| Expenditure Plan for Carryover Budget |
This sum (111,481 yen) is planned to be spent on the purchase of miscellaneous computer accessories and biochemical reagents necessary for cell-free protein synthesis.
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Research Products
(6 results)
