Analysis of Biological Domains in Insecticidal Protein from B. Thuringiensis

• Project/Area Number: 01560105
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: 応用生物化学・栄養化学
• Research Institution: University of Osaka Prefecture
• Principal Investigator: HIMENO Michio Univ. of Osaka Pref., College of Agric., Professor, 農学部, 教授 (10026411)
• Co-Investigator(Kenkyū-buntansha): WADANO Akira Univ. of Osaka Pref., College of Agric., Lecturer, 農学部, 講師 (40081575)
• Project Period (FY): 1989 – 1990
• Project Status: Completed (Fiscal Year 1990)
• Budget Amount: ¥2,100,000 (Direct Cost: ¥2,100,000); Fiscal Year 1990: ¥500,000 (Direct Cost: ¥500,000); Fiscal Year 1989: ¥1,600,000 (Direct Cost: ¥1,600,000)
• Keywords: Bacillus thuringiensis / Insecticidal protein / ICP / Domain / Protein engineering / delta-endotoxin / Deletion mutant / Bacillus thuriensis / Sー内毒素 / Bacillus thuringiensis

Research Abstract

Bacillus thuringiensis var. Israelensis (Bti) produces insecticidal crystalline protein (ICP), delta-endotoxin, which specifically kills dipteran insect larvae, mosquito and black fly. The ICP have several toxicities, that is, 1) dipteran insecticidal, 2) insect cell toxic, 3) insect specific neurotoxic, 4) mammalian cell toxic, 5) mammalian hemolytic and 6) suckling mouse toxic activities. The ICP senes encode 128 kDa (ISRH3) and 135 kDa (ISRH4) proteins and determined as respects the nucleotide sequences. The ISRH3 gene is consisted of 3405 by encoding 1135 amino acid. Then in this project, we attempt to identify the active domain in ISRH3 protein on the activities.
Several mutants of ISRH3 deleted from C of N terminal were constructed by treatment of several restriction enzymes and the proteins from the genes were assayed by the mosquito larvae and an established insect cell (TN-368 ). The protein produced by the gene encoding from N terminal to No. 635 amino acid has also insecticid al and insect cytolytic activities. However, the peptide from N terminal to No. 602 loses these two activities. The proteins of No. 613 - No. 1135 amino acid (C terminal) have the two activities, though the proteins of No. 632 or No. 641 - C terminal showed no clear results as to the activities. Then that protein of No. 716 - No. C terminal had lost the activities. The ISRH3 proteins deleted a central parts No. 560 - No. 772 amino acid had also lost its. The several small peptides central parts, No. 560 - No. 820 had lost the activities. From these results the central parts included No. 613 - No. 635 amino acid is one of domain and another domain (R or R') locate at N or C terminal, respectively. It was suggested that the domains for insecticidal and insect cell toxic activities required two domains, that is, A and another R or R' domains and also suggested the insecticidal activities was the insect cell toxic activity. However, it is ISRH3 does not have the mammalian cell toxic, hemolytic activities and the suckling mouse toxicity. The epitop of an antibody against ISRH proteins recognizes amino acid sequences around No. 773 - No. 820.