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  • 11
    Electronic Resource
    Electronic Resource
    Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis (1997)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 146 (1997), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The Bacteroides fragilis enterotoxin is an extracellular zinc metalloprotease that has been implicated in diarrheal disease of humans and animals. This toxin causes fluid accumulation in intestinal loops and is cytotoxic for HT-29 cells, an intestinal carcinoma cell line. Here we report the cloning and sequencing of the toxin gene (bftP). bftP is 1191 nucleotides coding for a 397 amino acid protein of 44.4 kDa. The toxin has a signal peptide of 18 amino acids that is typical of many lipoproteins followed by a 379 amino acid protoxin. The portion of the protoxin found in culture filtrates and stools begins at amino acid 212. An additional open reading frame located immediately upstream shows some sequence identity with cobra cytotoxins. If expressed, the ORF protein product could also play a role in the virulence of B. fragilis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1997.tb10205.x
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  • 12
    Electronic Resource
    Electronic Resource
    Cloning of the carbohydrate-binding portion of the toxin A gene ofClostridium difficile (1987)
    Springer
    Current microbiology 16 (1987), S. 55-60 
    Details
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A portion of the toxin A gene ofClostridium difficile was cloned into pBR322 withEscherichia coli Chi 1776 as the host. Five identical clones, each containing a 4.7-kbPstI restriction endonuclease fragment and producing toxin A antigens, were detected with affinity-purified, monospecific antibodies against toxin A. Digestion of the cloned DNA withPstI revealed as internal restriction site that resulted in two fragments (2.1 and 2.6 kb in size). Probe DNA from either of these fragments hybridized with DNA in the 4.7 kb region ofPstI-digested, high-molecular-weight DNA from the sourceC. difficile strain, indicating that the internalPstI site is protected. The probe DNA also hybridized with restriction-digested DNA from five additional toxigenic strains, but it did not hybridize with DNA from four nontoxigenic strains. In addition, a DNA fragment from a toxigenic strain ofClostridium sordellii, whose toxin cross-reacts with antibody toC. difficile toxin A, hybridized with the clonedC. difficile DNA. Unlike native toxin A, the cell lysate from the recombinant clone was not cytotoxic to Chinese hamster ovary cells or enterotoxic in hamsters. It did agglutinate rabbit red blood cells, a characteristic of toxin A. The cell lysate also exhibited a line of partial identity when compared with purified toxin A in Ouchterlony assays, and it reacted with monoclonal antibody to toxin A in an enzyme-linked immunosorbent assay. The cloned DNA appears to code for a nontoxic binding portion of toxin A, which is responsible for binding to galactose-α1-3-galactose-β1-4-N-acetylglucosamine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01568170
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  • 13
    Electronic Resource
    Electronic Resource
    Vaccination against lethalClostridium difficile enterocolitis with a nontoxic recombinant peptide of toxin A (1990)
    Springer
    Current microbiology 21 (1990), S. 29-32 
    Details
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Toxin A ofClostridium difficile has a complex series of repeating units, each 20 or 30 amino acids in length, located at the COOH-terminus of the molecule. In the following study, we found that antiserum against a nontoxic recombinant peptide comprising 33 of the 38 repeating units neutralized the enterotoxic and cytotoxic activity of the toxin and that hamsters vaccinated with the recombinant peptide were partially protected againstC. difficile disease.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02090096
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  • 14
    Electronic Resource
    Electronic Resource
    Nonspecific binding of mouse monoclonal antibodies toClostridium difficile toxins A and B (1989)
    Springer
    Current microbiology 19 (1989), S. 303-306 
    Details
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Several recent reports have described large numbers of monoclonal antibodies that cross-react with toxins A and B ofClostridium difficile; this suggests that the toxins share major epitopes. Our results show that monoclonal antibodies (MAb) against other antigens bind nonspecifically to both toxins. Therefore, we believe that the cross-reacting MAb bind by this manner and not by a true immune reaction.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01570105
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  • 15
    Electronic Resource
    Electronic Resource
    Cloning and expression of the toxin B gene ofClostridium difficile (1990)
    Springer
    Current microbiology 20 (1990), S. 397-401 
    Details
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5′-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02095867
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  • 16
    Electronic Resource
    Electronic Resource
    Isolation of recombinant proteins from milk (1992)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 49 (1992), S. 333-338 
    Details
    ISSN: 0730-2312
    Keywords: Casein micelle ; Immunopurification ; Lactation ; Monoclonal antibody ; Post-translational modification ; proteases ; therapeutics ; viral inactivation ; whey ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Milk is a complex bio-colloid which presents some unique problems for the protein isolation chemist, but the majority of the processing criteria for purifying recombinant proteins are the same as with any complex biological mixture. The casein micelles and fat globules behave as separate phases; they prevent filtration of the milk and interfere with the usual separation methods. The usual first step is to centrifuge the milk to remove the fat and precipitate the casein micelles with low pH or precipitating agents. Some recombinant proteins may associate to some degree with the micelles which may necessitate solubilizing them with chelating agents. If the majority of the product protein associates with either the fat or micelles, this can be used to advantage. Once the casein micelles have been removed or disrupted, the clarified milk can be processed by the usual separation methods. There also are proteases in milk which can degrade recombinant proteins. The greatest advantage of producing recombinant proteins in milk is the high concentration which can be obtained. The high levels of product protein can alleviate many problems associated with the application of classical purification strategies to transgenic milk proteins.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240490403
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