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

Abstract

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...

The 42- and 51-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: construction of recombinants with enhanced expression and in vivo studies of processing and toxicity

“…Oeda et al (26) reported that when E. coli contained a plasmid with the gene for the 8 endotoxin of B. thuringiensis var. aizawai, temperature determined the appearance of the inclusions.…”

Specific Toxicity of δ-Endotoxins fromBacillus thuringiensistoBombyx mori

“…aizawai which were produced and accumulated in the recombinant Escherichia coli JMI03. 21 ) The inclusions contained CryIA(a) or CryIA(b) in almost the same amount (Fig. 1, lanes 2 and 3).…”

Cytolytic activity of Bacillus thuringiensis CryIC and CryIAc toxins to Spodoptera sp. midgut epithelial cells in vitro

“…The CrylC e~stals produced from the E. coli recombinant at 37 ° C were predonfinantly amorphous shaped (not shown), possibly due to temperature effects on protein assembly (29). Other groups also have documented the presence of small peptides associated with various toxins expressed in recombinant E. coil (10,27,29). Eater fractions contained CryIC purified to a single protein band as determined by SDS-PAGE.…”

“…The CrylC e~stals produced from the E. coli recombinant at 37 ° C were predonfinantly amorphous shaped (not shown), possibly due to temperature effects on protein assembly (29). The CrylC e~stals produced from the E. coli recombinant at 37 ° C were predonfinantly amorphous shaped (not shown), possibly due to temperature effects on protein assembly (29).…”