2001 Fiscal Year Final Research Report Summary
Protein engineering of a plant bioactive protein karasurin.
| Project/Area Number |
10671993
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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 |
Chemical pharmacy
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| Research Institution | Nagoya City University |
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Principal Investigator |
MIZUKAMI Hajime Nagoya City University, Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (30128219)
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| Project Period (FY) |
1998 – 1999
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| Keywords | Ribosome-inactivating activity / RNA N-glycosidase / Karasurin / Recombinant protein / Protein engineering |
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| Research Abstract |
Karasurin is a basic protein accumulated in root tubers of Trichosanthes kirilowii var. japonica, and exhibit ribosome-inactivating and abortifacient activities. This project has bee carried out to analyze the relationship between amino acid sequence of karasurin and its ribosome-inhibiting activity using the protein engineering approach. Following results have been obtained to date.
1. cDNA clones encoding karasurin was isolated and characterized.
2. An expression vector to produce recombinant karasurin as a fusion protein with maltose-binding protein (MBP) was constructed and over-expression system using E. coli was established.
3. The recombinant fused protein was recovered in soluble fraction and highly purified by amylose-resin column chromatography. Karasurin was obtained by factor Xa treatment of the fused protein.
4. For the assay methods to evaluate ribosome-inactivating activity, both HPLC assay to detect adenine molecule from rRNA and electrophoretic assay to detect a fragment released from 28S-rRNA were established.
5. By using these assay methods following points were clarified.
(1) Recombinant karasurin fuse with MBP showed the same level of ribosome-inactivating activities as native karasurin.
(2) Modified karasurin in which C-terminal 20 amino acids were truncated lost its ribosorme-inactivating activity. This shows that C-terminal sequence of karasurin is important to express the ribosome-inactivating activity.
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