苏云金杆菌4.0718菌株杀虫晶体65kD原毒素的质谱分析

以苏云金杆菌4.0718菌株杀虫晶体65kD原毒素为材料,探索了从SDS－PAGE胶上回收蛋白质进行质谱分析的方法。蛋白纯化的步骤包括SDS－PAGE、电洗脱、脱盐、除SDS。电洗脱采用半透膜法,用超滤法脱盐,冷丙酮沉淀法除去SDS。结果表明,纯化的65kD原毒素经ESI－MS检测,分子量约64500Da。经MALDI－MS检测,未能有明显蛋白峰出现,这可能是该蛋白由于较强的疏水作用,溶解性差,在与基质混合时处于聚和悬浮态所致。     

Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera)

The midgut proteases of the Bacillus thuringiensis resistant and susceptible populations of the diamondback moth, Plutella xylostella L. were characterized by using protease specific substrates and inhibitors. The midgut contained trypsin-like proteases of molecular weights of 97, 32, 29.5, 27.5, and 25 kDa. Of these five proteases, 29.5 kDa trypsin-like protease was the most predominant in activation of protoxins of Cry1Aa and Cry1Ab. The activation of Cry1Ab protoxin by midgut protease was fast (T(1/2) of 23-24 min) even at a protoxin:protease ratio of 250:1. The protoxin activation appeared to be multi-step process, and at least seven intermediates were observed before formation of a stable toxin of about 57.4 kDa from protoxin of about 133 kDa. Activation of Cry1Aa was faster than that of Cry1Ab on incubation of protoxins with midgut proteases and bovine trypsin. The protoxin and toxin forms of Cry proteins did not differ in toxicity towards larvae of P. xylostella. The differences in susceptibility of two populations to B. thuringiensis Cry1Ab were not due to midgut proteolytic activity. Further, the proteolytic patterns of Cry1A protoxins were similar in the resistant as well as susceptible populations of P. xylostella.     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices 1 and 2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the 1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.     

Assessment of protoxin composition of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by use of polyacrylamide gel block and mass spectrometry

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zujiao Fu and Yunjun Sun contributed equally to this work.     

The main features of Bacillus thuringiensis δ-endotoxin molecular structure

The crystal-forming proteins (-endotoxins) produced by various serotypes of Bacillus thuringiensis and toxic for Lepidoptera reveal the same pattern of molecular organisation. These proteins (M _r of ca. 145,000–130,000) contain an N-terminal domain (M _r of 85,000–65,000) resistant to proteolysis whereas their C-terminal moieties (M _r of 65,000) undergo an extensive degradation by trypsin that leads to stepwise cleavage off the fragments with M _r of 15,000–35,000.The N-terminal domain from serotype V -endotoxin is active when introduced into the hemocoel of Galleria mellonella larvae. Hence, it correponds to the true toxin normally formed by larva proteases action on the crystalforming protein (protoxin). Some differences were found in the properties of the N-terminal domains isolated from the crystal-forming proteins of III, V and IX serotypes, e.g., in their solubility, digestion by subtilisin, molecular weights and the distribution of methionine residues along the polypeptide chains.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacryl amide gel electrophoresis - CFP crystal-forming protein - DNS 5-dimethylamino-1-naphthalene-sulphonyl     

Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involves palmitoylation of lysine 983 and is dependent on co-expression of cyaC. The role of the cyaC gene product in the acylation reaction has not been determined. We have developed an efficient T7 RNA polymerase system for over-expression of cyaA and cyaC separately in Escherichia coli. Each protein accumulated intracellularly in an insoluble form and could be collected by centrifugation of lysed cells. A single-step purification was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCyaA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cytosolic factor required for toxin activation was lost if the S100 extract was dialysed before use and the cytosolic factor could be replaced in the in vitro reaction by ACP charged separately in vitro with palmitic acid, as reported previously for activation of the homologous E. coli haemolysin (HIyA). The in vitro activation system may be used to investigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target cell specificity and toxic activity of CyaA     

Proteolytic Cleavage of Tetanus Toxin Increases Activity

Tetanus toxin is initially synthesized in the form of a single polypeptide chain and then proteolytically "nicked" by the bacteria to produce a two-chain structure joined by a disulfide bond. This two-chain form of the toxin is the form known to be biologically active. Whether such nicking is necessary for activity, as it is for certain other bacterial toxins, has not been demonstrated previously. Single-chain toxin preparations produced by salt extraction from the bacteria are characterized and compared with pure two-chain toxin obtained from extracellular filtrates. The ability of these various toxin preparations to produce paroxysmal activity in mouse spinal cord neurons grown in dissociated cell culture is described. The pure two-chain toxin is demonstrated to have greater activity than the single-chain toxin preparations. Indeed the activity of the single-chain toxin preparations can be explained by the small amounts of residual two-chain toxin present in these extracts. Using a protease from a toxin-minus strain of Clostridium tetani to convert a single-chain toxin preparation to two-chain toxin increases toxin activity. In vivo the single-chain toxin preparation is also less toxic. These findings indicate that proteolytic nicking of tetanus toxin increases activity. The unnicked, single-chain form of tetanus toxin may be a relatively nontoxic protoxin form of the toxin; this is a structure-function relationship similar to that of other bacterial protein toxins.     

Processing of Cry1Ab δ-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices α1 and α2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the α1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.