Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli

Oeda, Kenji; Oshie, Kazuyuki; Shimizu, Masatoshi; Nakamura, Keiko; Yamamoto, Haruyasu; Nakayama, Isamu; Ohkawa, Hideo

doi:10.1016/0378-1119(87)90098-9

Cited by 46 publications

(36 citation statements)

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“…Expression of several cryI genes in E. coli and B. subtilis leads to the synthesis of biologically active inclusions (14,16,46,50), suggesting that the 130-to 140-kDa CryI proteins can spontaneously form crystals independent of the host bacterium. These results appear to agree with the principle of protein self-assembly, as first suggested by Lecadet's observation (34) that bipyramidal crystals denatured in 8 M urea reverted to their original shape when the urea was removed by dialysis.…”

Section: Crystallization Of the Insecticidal Proteinsmentioning

confidence: 99%

How does Bacillus thuringiensis produce so much insecticidal crystal protein?

1995

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Section: Crystallization Of the Insecticidal Proteinsmentioning

confidence: 99%

How does Bacillus thuringiensis produce so much insecticidal crystal protein?

1995

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“…2A). In Western blot (immunoblot) experiments, degraded IP bands were not observed with the cell extracts of E. coli JM103(pTB1) (14).…”

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“…aizawai IPL7 exhibits insecticidal activity toward lepidopteran larvae and synthesizes mainly 130-and 135-kilodalton (kDa) insecticidal proteins (IPs) which form bipyramidal crystals. The two IP-encoding genes were cloned, and the primary structures were deduced from their nucleotide sequences (14,15 Analysis of amorphous and crystallike bodies formed in E. coli by SDS-PAGE. After E. coli JM103(pTB1) cells were cultured at 37 and 30°C, both amorphous and crystallike bodies were separately prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 11).…”

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Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli

et al. 1989

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Escherichia coli JM103 cells harboring expression plasmid pTB1 or pKC6 synthesized the 130-and 135-kilodalton insecticidal proteins, respectively, of BaciUus thuringiensis subsp. aizawai IPL7, and both products accumulated as cytoplasmic inclusion bodies. Amorphous inclusions which contained contaminating proteins, together with the corresponding insecticidal proteins, were formed in cultures at 37°C, but bipyramidal crystals practically free of contaminants were observed at 30°C. Although 9.8% of the amino acids were substituted between these two proteins, both protein crystals had the same shape as those of the parental B. thuringiensis strain, which produced both proteins.Bacillus thuringiensis produces proteinaceous crystals toxic to insect larvae (5,7,10). Depending on the subspecies, the crystals are specifically toxic to lepidopteran (8, 9), dipteran (12,16,17), or coleopterous (2, 4, 6) larvae. B. thuringiensis subsp. aizawai IPL7 exhibits insecticidal activity toward lepidopteran larvae and synthesizes mainly 130-and 135-kilodalton (kDa) insecticidal proteins (IPs) which form bipyramidal crystals. The two IP-encoding genes were cloned, and the primary structures were deduced from their nucleotide sequences (14,15 37°C in L broth medium (13) were examined for inclusion bodies by phase-contrast microscopy (Fig. 1A). Distinct inclusion bodies were first observed after 8 h of incubation. These continued to grow until the late exponential growth phase and finally reached a length exceeding 0.5 ,um (Fig. 1A). At the stationary growth phase, over 95% of the E. coli cells contained the inclusion bodies. No phase-refractile inclusions were found in the negative control cells of E. coli JM103(pKK223-4) (data not shown). The inclusion bodies were easily recovered from the cells by a combination of freeze-thawing, sonication, and centrifugation (4,500 x g, 5 min). Under microscopic observation, the size and shape of the inclusion bodies were not uniform (Fig. 1B). When E. coli JM103(pTB1) cells were cultured at 30°C, crystallike inclusion bodies were formed (Fig. 1C) and most were of much the same size (Fig. 1D) Analysis of amorphous and crystallike bodies formed in E. coli by SDS-PAGE. After E. coli JM103(pTB1) cells were cultured at 37 and 30°C, both amorphous and crystallike bodies were separately prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 11). The main protein band in the amorphous bodies was the 130-kDa IP, although other protein bands with lower molecular masses were found ( Fig. 2A). In Western blot (immunoblot) experiments, degraded IP bands were not observed with the cell extracts of E. coli JM103(pTB1) (14).Several washes of the inclusion bodies with 0.5 M NaCl gave similar results, indicating that the lower-molecular-mass proteins were involved in the inclusion bodies. ADC-18 densitometer (Gelman Sciences, Inc.) scanning of the gel indicated that the purity of the 130-kDa IP was about 70%. The crystallike bodies contained lesser amounts of other small p...

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“…6 ) We cloned a 130-kDa IP gene from B. thuringiensis subsp. aizawai IPL7 7 ) and expressed it in Escherichia coli cells. The 130-kDa IP produced in the recombinant E. coli cells was processed in gut juice of Spodoptera litura larvae and yielded a protease-resistant fragment with a molecular mass of 60 kDa, which was insecticidally active.…”

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