2012 Fiscal Year Final Research Report
Novel screening protocol for multi-SS bond proteins using SEP tags and its application to the development of a minimal Luciferase
| Project/Area Number |
23651213
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Living organism molecular science
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| Research Institution | Tokyo University of Agriculture and Technology |
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Principal Investigator |
KURODA Yutaka 東京農工大学, 大学院・工学研究院, 准教授 (10312240)
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| Project Period (FY) |
2011 – 2012
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| Keywords | SS結合ミスマッチ / 折り畳み / 溶解度向上タグ / 可溶化タグ / 逆相HPLC / 天然状態 / 生物発光 / SEPタグ |
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| Research Abstract |
Bioluminescent proteins are indispensable tools for bio-imaging, and controlling and manipulating their molecular properties has become an important issue in protein engineering. Here we developed a novel screening method for improving the bioluminescent properties of Gaussia luciferase (GLuc). GLuc possesses 10 cysteins forming 5 SS-bonds, which make GLuc manipulation especially difficult, because the cysteins become entangled into non-native SS-bonds when GLuc is expressed in E-coli with traditional protocols. In the present study we used SEP tags to solubilize the protein and favor the formation of native SS-bonds in E-coli, which is a handy and inexpensive expression host. Using this new technique we isolated novel GLuc variants with red-shifted luminescence. The application range of this method can be extended to other SS-bond containing proteins, and the improved GLuc will contribute to complement the register of reporter proteins.
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