| Project/Area Number |
16J09051
|
|---|
| Research Category |
Grant-in-Aid for JSPS Fellows
|
| Allocation Type | Single-year Grants |
|---|
| Section | 国内 |
|---|
| Research Field |
Veterinary medical science
|
|---|
| Research Institution | Kyoto Sangyo University |
|---|
Principal Investigator |
TONITI Waraphan (2017) 京都産業大学, 工学研究科, 特別研究員(DC2)
TONITI WARAPHAN (2016) 京都産業大学, 工学研究科, 特別研究員(DC2)
|
|---|
| Project Period (FY) |
2016-04-22 – 2018-03-31
|
| Project Status |
Completed (Fiscal Year 2017)
|
| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2017: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2016: ¥700,000 (Direct Cost: ¥700,000)
|
|---|
| Keywords | enterotoxin / food-poisoning / Clostridium / binary toxin / CPILE / x-ray crystallography |
|---|
| Outline of Annual Research Achievements |
The W5052 strain isolated from unusual Tokyo food-poisoning outbreaks did not harbor the cpe nor produce C. perfringens enterotoxin (CPE). On the other hand, they secreted a novel enterotoxin, CPILE. It is consisting of two components: CPILE-a, which acts as an enzymatic ADP-ribosyltransferase and CPILE-b, a membrane binding component.
The enterotoxicity of CPILE relies on endocytosis of the CPILE complex into the cytosol. CPILE-b binds to the specific cell receptor and assists translocation of CPILE-a into the target cell. How CPILE-b binds to the particular cell receptor is still unclear.
The purified CPILE-b was subjected to cleavage pro-sequence region by trypsin to trigger oligomerization. It should be noted that the SDS-PAGE of the purified CPILE-b showed both pro-sequence and CPILE-b bands. Then, the purified CPILE-b was concentrated and crystalized using standard crystallization kits via hanging drop vapor diffusion method. CPILE-b crystals found after one month screening and SDS-PAGE of selected crystals confirmed that they were CPILE-b crystals. However, poor data set was collected and failed to assign the correct space group even though the good shapes of CPILE-b crystals were used. The pro-sequence probably nicked to CPILE-b and disturbed good quality of crystal formation.
In conclusion, N-terminal GST-tag CPILE-b with regular purification procedure failed to separate pro-sequence region from CPILE-b body. The condition that triggers oligomerization of CPILE-b should be further investigation.
|
| Research Progress Status |
29年度が最終年度であるため、記入しない。
|
| Strategy for Future Research Activity |
29年度が最終年度であるため、記入しない。
|
