1999 Fiscal Year Final Research Report Summary
Development of the system for a large-scale expression of membrane proteins towards their crystallization.
| Project/Area Number |
10680632
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Nagoya University |
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Principal Investigator |
SUGIYAMA Yasuo Nagoya University, Institute for Gene Research, Associate Professor, 遺伝子実験施設, 助教授 (70154507)
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| Co-Investigator(Kenkyū-buntansha) |
IHARA Kunio Nagoya University, Institute for Gene Research, Assistant Professor, 遺伝子実験施設, 助手 (90223297)
KUSUMI Akihiro Nagoya University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (50169992)
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| Project Period (FY) |
1998 – 1999
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| Keywords | Retinal proteins / light-driven ion pump / heterologous expression ysytem / fission yeast / GFP-fusion protiens / two-dimensional crystal / tubular structure |
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| Research Abstract |
We have examined the methods to isolate large amounts of intact retinal proteins expressed in Schizoseaccharomyces pombe. Retinal proteins with (His)6-Tag at a C-terminal were expressed in S.pombe, however, they could not be purified by Ni-NTA agarose column chromatography. Therefore, it was concluded that (His) 6-Tag method does not replace the conventional one.
Retinal proteins with GFP at a C-terminal were expressed and distributed in the membranes of ER and vacuole in S.pombe at a log phase. At a stationary phase, they were digested and released GFP were distributed in cytoplasm and nuclear membrane. Therefore, it is necessary to disrupt the cells immediately after expression the proteins.
We found that archaerhodopsin (aR) forms two-dimensional crystal in the membrane as bacteriorhodopsin does and that aR transforms from membrane sheet to tubular structure when stored in a refrigerator. We are going to find out the condition to crystallize aR.
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