| Project/Area Number |
17370043
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | National Institute of Advanced Industrial Science and Technology |
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Principal Investigator |
ISHII Noriyuki National Institute of Advanced Industrial Science and Technology, Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Senior Research Scientist (10261174)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,580,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2007: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2006: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 2005: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Keywords | Complement receptor / sCR1 / C3b / C4b receptor / CHO cell / sCR1 |
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| Research Abstract |
Although the application of animal cells to artificial organ in the field of organ-therapy has become an active and established industry, various problems arise during xenograft. One of the most difficult problems is the immune reaction caused by complement response. In this study we have developed a more efficient method to purify sCR1 (soluble complement receptor type I) which can be used to suppress the complement activation and prevent inflammation during xenograft. We have reported a successful observation by TEM of sCR1 prepared according to our novel purification method. The CHO cells are used to produce human sCR1. Our purification method consists of a two-stage cell culture (cultivating cells in serum medium followed by serum-free medium), and a two-stage column purification by means of heparin and gel filtration. The activity of sCR1 is analyzed by the reaction with factor I and C3ma. Morphology of sCR1 was examined by TEM (Philips Tecnai F20). For the two-stage purification, the heparin column not only bound sCR1 selectively but also condensed sCR1 to a concentration 10 fold (400ug/ml) higher than that obtained from cell supernatant (40ug/ml). HPLC gel filtration column chromatography showed a single peak, and SDS-PAGE indicated only one band close to 220 kDa, suggesting high purity of sCR1. The amount yield was 10-20 mg/1 L culture. The co-factor activity assay showed that sCR1 retained its activity. In EM, sCR1 molecule mono-dispersed in an appropriate surfactant was found to be square-shaped with a side length of 11.6 nm. More of trials of crystallization for sCR1 are under further investigation.
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