• Journal of Food Hygiene and Safety
• /
• v.3 no.2
• /
• pp.75-81
• /
• 1988

Various methods such as lel-mtration on Sephadex G-SO, 75, 100 Sephacry, and Ultro gel, and lon-exchanle chromatoaraphy on Amberilte and carboxymethyl (CM)-cellulose, and Fast Protein liquid Chromatolraphy (FPLC) were applied for the purification of enterotoxin B from Staphylococcus aureus ATCC 14458 and compared one another. lon-exchanle chromatography on Amberllte resin was good enough to remove non-entrotoxln materials in culture, convlnient to use and fast although tbe purity was less tban 70%. However, CM-cellulose showed to be better purity and yield tban those of Amberilte resin. The yields of these two resins for ion-exchange cbromatograpby were about 70% and 75%, respectively. When the gel-filtration methods on Sepbadex G-50, 75, 100, Sepbacryl, and Ultro lei were applied, the purities were about 90%. FPLC was found to be tbe most efficient metbod in terms of purity (96%) and speed. For the purification of sample with large volume, particularly, tbe combined metbod, gel-mtration after Amberlite can be also used efficiently. Tbe purified toxin was found to be identical to type B enterotoxin used for reference standard by Oucbterlony immunodiffusion test.

• Journal of Plant Biotechnology
• /
• v.6 no.1
• /
• pp.21-24
• /
• 2004

A modified $\delta$-endotoxin gene of Bacillus thuringiensis ssp. tenebrionis (B.t.t.), encoding a coleoptera-specific toxin, was utilized to transform citrus plants, Citrus reticulata Blanco 'Ponkan' mandarian. By co-culturing the nucelli with Agrobacterium tumefaciens harboring the modified gene in the binary vector pBinAR-Btt, the chimeric toxin gene was transferred into citrus plants. The transgenic plants were selected on modified Murashige and Skoog medium containing kanamycin. Hybridization experiments demonstrated that the transgenic plants contained and expressed the toxin protein gene.

• Korean Journal of Veterinary Research
• /
• v.47 no.1
• /
• pp.59-65
• /
• 2007

Pasteurella multocida Pasteurella multocida toxin (PMT) is a causal pathogenin atrophic rhinitis in pigs. To investigate the protective immunity and vaccination effect of recombinantPMT, the gene for PMT was isolated from the infective P. multocida D:4. The 2.3 kb XhoI/PstI fragment(PMT2.3) of PMT gene was expressed in E. coli BL21 (DE3) using the induced expression vector system.The recombinant protein of PMT2.3 having molecular weight of 84 kDa was purified by Ni-afinitycolumn chromatography. The PMT2.3 raised slightly less anti-PMT antibody titer than formalin-killedwhole cel, however, it showed more protective imunity against P. multocida D:4 infection in vaccinationand chalenge.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.6
• /
• pp.788-792
• /
• 2012

We report the computational structural simulation of the Cry1Ab19 toxin molecule from B. thuringiensis BtX-2 based on the structure of Cry1Aa1 deduced by x-ray diffraction. Validation results showed that 93.5% of modeled residues are folded in a favorable orientation with a total energy Z-score of -8.32, and the constructed model has an RMSD of only $1.13{\AA}$. The major differences in the presented model are longer loop lengths and shortened sheet components. The overall result supports the hierarchical three-domain structural hypothesis of Cry toxins and will help in better understanding the structural variation within the Cry toxin family along with facilitating the design of domain-swapping experiments aimed at improving the toxicity of native toxins.

• BMB Reports
• /
• v.53 no.12
• /
• pp.611-621
• /
• 2020

Bacterial endoribonuclease toxins belong to a protein family that inhibits bacterial growth by degrading mRNA or rRNA sequences. The toxin genes are organized in pairs with its cognate antitoxins in the chromosome and thus the activities of the toxins are antagonized by antitoxin proteins or RNAs during active translation. In response to a variety of cellular stresses, the endoribonuclease toxins appear to be released from antitoxin molecules via proteolytic cleavage of antitoxin proteins or preferential degradation of antitoxin RNAs and cleave a diverse range of mRNA or rRNA sequences in a sequence-specific or codon-specific manner, resulting in various biological phenomena such as antibiotic tolerance and persister cell formation. Given that substrate specificity of each endoribonuclease toxin is determined by its structure and the composition of active site residues, we summarize the biology, structure, and substrate specificity of the updated bacterial endoribonuclease toxins.

• Journal of Life Science
• /
• v.29 no.1
• /
• pp.97-104
• /
• 2019

Streptococcus iniae is a typical fish pathogen causing streptococcosis and it can also cause zoonotic infectious diseases. We studied S. iniae FP5228 isolated from infected olive flounder in Wando, Korea. In a study to find virulence factors in FP5228, we found that the number of live bacteria decreased dramatically in culture medium containing S. iniae FP5228 for more than 24 hr. This phenomenon was hypothesized to be related to Toxin ${\zeta}$ and Antitoxin ${\varepsilon}$ genes, components of the Toxin/ Antitoxin (TA) system on the 14 kb plasmid of FP5228. We used a protein overexpression system to identify it. The pBP1140 vector system was constructed to regulate the expression of Toxin ${\zeta}$ and Antitoxin ${\varepsilon}$ by IPTG and Arabinose. E. coli/pBP1140 strain grew slowly in early growth under toxin expression condition, and it was confirmed by microscopic observation that the strain became longer. S. iniae CK287, lacking a 14 kb plasmid of S. iniae FP5228 strain, was constructed. CK287 bacterial cells did not show rapid killing during culture, and the ability to produce biofilm was also decreased, and toxicity was weakened in cytotoxicity test and fish test. These results suggest that the TA system is involved in physiological regulation and expression of virulence factors in S. iniae FP5228.

• Korean Journal of Food Science and Technology
• /
• v.20 no.6
• /
• pp.780-786
• /
• 1988

In order to investigate the most efficient and rapid method for the purification of enterotoxin A from Staphylococcus aureus M 7/1, various methods such as ion-exchange chromatography on Amberlite, and CM-cellulose. gel filtration on Sephadex G-50, 75, 100 and Sephacryl, and fast protein liquid chromatography (FPLC) were applied and compared in terms of purity and speed. Although ion-exchange chromatography on Amberlite resin was good enough to remove other materials in culture medium from enterotoxin, and convenient, and fast method, the purity of this method was less than 70%. However. carboxymethyl ion-exchange column showed to be better purity than that of Amberlite method. The yields of these two methods were about 70% and 75%, respectively. When gel filtration methods on Sephadex G-50, 75, 100 and Sephacryl were applied, the purity was about 90%. Fast protein liquid chromatography was found to be the most efficient method in terms of purity (97%) and speed. The combined method, gel filtration after CM-cellulose column (stepwise elution) treatment can be also used as a efficient method particularly for the purification of large volume of sample.

• KSBB Journal
• /
• v.22 no.1
• /
• pp.48-52
• /
• 2007

This study was carried out to establish the optimum disruption condition of a sonificator for the protein toxin for the purpose of developing automatic biological agent detector equipped a sonificator. One of the best-known collisional quenchers is molecular oxygen, which quenches almost all known fluorophores. The sonification does an excellent job of degassing, which decreased the quenching effect and increased the fluorescence quantity. The fluorescence measurement for the protein using 0.7 X fluorescent dye concentration and above must be done in 1 minute and the fluorescence measurement for the protein using 0.3 X fluorescent dye concentration and below has to be done between 2 and 3 minute. The fluorescence quantity of the sonificatied protein sample was much higher that of the non-sonificatied protein sample. Sonificating the sample turned out to be favorable for the fluorescence measurement when measuring at the low protein concentration.

• Biomolecules & Therapeutics
• /
• v.11 no.3
• /
• pp.157-162
• /
• 2003

The generation of secretory IgA antibodies(Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimHIctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was analyzed. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/CTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of CTXB. This study also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked CTXA2B acts as a useful mucosal adjuvant, and that the adhesin/CTXA2B chimeric protein might be a potential antigen for oral immunization against UPEC.

• Journal of Periodontal and Implant Science
• /
• v.38 no.sup2
• /
• pp.317-324
• /
• 2008

Purpose: Aggregatibacter actinomycetemcomitans was associated with localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis. The cytolethal distending toxin (CDT) of A. actinomycetemcomitans was considered as a key factor of these diseases is composed of five open reading frames (ORFs). Among of them, An enzymatic subunit of the CDT, CdtB has been known to be internalized into the host cell in order to induce its genotoxic effect. However, CdtB can not be localized in host cytoplasm without the help of a heterodimeric complex consisting of CdtA and CdtC. So, some studies suggested that CdtC functions as a ligand to interact with GM3 ganglioside of host cell surface. The precise role of the CdtC protein in the mechanism of action of the holotoxin is unknown at the present time. The aim of this study was to generate recombinant CdtC proteins expression from A. actinomycetemcomitans, through gene cloning and protein used to investigate the function of Cdt C protein in the bacterial pathogenesis. Materials and Methods: The genomic DNA of A. actinomycetemcomitans Y4 (ATCC29522) was isolated using the genomic DNA extraction kit and used as template to yield cdtC genes by PCR. The amplifed cdtC genes were cloned into T-vector and cloned cdt C gene was then subcloned to pET28a expression vector. The pET28a-cdtC plasmid expressed in BL21 (DE3) Escherichia coli system. Diverse conditons were tested to opitimize the expression and purification of functional CdtC protein in E. coli. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and comfirmed the recombinant CdtC expression by SDS-PAGE and Western Blotting. The expression level of the recombinant CdtC was about 2% of total bacterial proteins. Conclusion: The lab condition of procedure for the purification of functionally active recombinant CdtC protein is established. The active recombinant CdtC protein will serve to examine the role of CdtC proteins in the host recognition and enzyme activity of CDT and investigate the pathological process of A. actinomycetemcomitans in periodontal disease.