ISOLATION AND ANALYSIS OF RECEPTOR FOR INSECTICIDAL PROTEIN, delta-ENDOTOXIN
| Project/Area Number |
04660093
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用生物化学・栄養化学
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| Research Institution | University of Osaka Prefecture, College of Agriculture |
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Principal Investigator |
HIMENO Michio Univ.of Osaka Pref., College of Agric., Professor, 農学部, 教授 (10026411)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIMOTO Kenji Univ.of Osaka Pref., College of Agric., Assistant, 農学部, 助手 (40196746)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1993: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1992: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Biopesticide / Bacillus thuringiensis / Receptor / Mid-gut, silkworm / BBMV / delta-Endotoxin / 糖タンパク質 / delta-内毒素 / 殺虫性タンパク質 / S-内毒素 |
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| Research Abstract |
The delta-endotoxin produced by Bacillus thuringiensis was known as a crystalline protein body (CPB). This CPB was also used as microbial pesticide. It seem that a factor to determine the specific toxicity of CPB is a receptor in brush border membrane (BBM) of mid-gut epithelial cells. Then in this project, we attempt to isolation and analysis of the receptor from BBM of the silkworm mid-gut for {CryIA(a)}.
An insecticidal protein, CryIA (a) was isolated from E.coli carrying CryIA (a) gene cloned from B.thuringiensis var.aizawai. In order to isolate the receptor for CryIA (a), the brush border membrane vesicle (BBMV) was solubilized by 2% cholic acid, and was adsorbed on the CryIA (a) -immobilized Tresyl Toyopearl affinity column. The eluate from the affinity column was applied on a DEAE-Toyopearl. The binding activities with ^<125>I-CryIA (a) of the purified receptor by the DEAE-column were determined and blocking activities of ^<125>I-CryIA (a) against BBMV or solubilized BBMV were al
… More
so detected. An electropherogram of the receptor protein on PAGE was determined by the binding activity with ^<125>I-CryIA (a). A main 180 kDa and minor 170 kDa bands were detected on the autoradiogram of ^<125>I.The 170 kDa band was removed by using of proteinase inhibitors in all purification processes. It was confirmed that the only 180 kDa protein was a receptor protein by the competition experiments of ^<125>I-CryIA (a) and BBMV or the solubilized BBMV.The 180 kDa protein was not a leucine aminopeptidase or an alkaline phosphatase. It was determined that the molecular weight of the native receptor protein was about 600 kDa by a gel filtration column chromatography. These results suggested that the native receptor protein was assemble by three 180 kDa protein. The N-terminal of 180 kDa protein was blocked by some residues, then the 180 kDa protein treated by a lysylendopeptidase and subsequently 6 oligopeptides were isolated from the digested solution by a HPLC.The N-terminal amino acid sequences of the oligopeptides were determined by a protein sequencer (Shimazu PSQ-1). The 180 kDa protein was a kind of glycosyl protein. Less
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Report
(4 results)
Research Products
(8 results)
