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  • 1
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The 5C outer membrane protein, one of the N. meningitidis class 5 proteins, was preferably expressed in bacteria isolated from the nasopharynx and its role in adhering to the mucosal cells and invading them as well as the development of anti-5C antibodies in healthy carriers was demonstrated. Anti-5C monoclonal antibodies are bactericidal in the presence of the human complement. The immunodominant region of the 5C protein is highly conserved among the different strains of N. meningitidis, and the opc gene, which encodes the protein, does not seem to show antigenic variations.Here the isolation of the opc gene from the Cuban strain B:4:P1.15 by PCR (Polymerase Chain Reaction) is presented. Under the regulation of the tryptophan promoter, the gene was cloned and sequenced in E. coli with a high level of expression and fused to the amino-terminal end of the interleukin-2 gene. In the dot-blot experiments, the presence of the gene in those strains which did not express the protein in the whole cell ELISA was also detectable.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The gene coding for the class 1 outer membrane protein from the Neisseria meningitidis strain B385 (B : 4 : P1.15) was isolated by the Polymerase Chain Reaction (PCR) and cloned into the Sma I cut M13mp 18 vector. Then, a Xba I restriction site was created by using an oligonucleotide-directed in vitro mutagenesis system at the start of the coding fragment. In order to express the protein, this fragment was fused to 180 base pairs corresponding to 60 amino acids from the N terminus of interleukin-2 under the control of the tryptophan promoter (Ptrp). The expression was confirmed by Western-blotting where the recombinant protein (PILM28) was detected by bactericidal monoclonal antibodies (MABs). The recombinant polypeptide was partially purified and used to elicit murine antibodies, able to recognize the meningococcal class 1 subtype 15.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: The Thermus aquaticus DNA Polymerase I gene was isolated by using standard recombinant DNA techniques and cloned into an E. coli vector in front of the lac promoter. E. coli cultures produced thermostable polymerase when induced with the analog IPTG. A purification procedure is shown which renders an enzymatic preparation suitable for PCR reactions.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0138-4988
    Keywords: Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cell disruption studies for the extraction of HBsAg from a recombinant P. pastoris strain (r-HBsAg) were done using a bead mill disintegrator. Three sequential passages (4 min retention time each) were enough to disrupt the cells and extract most of the r-HBsAg and soluble proteins. An acid precipitation step was performed just after cell disruption to precipitate proteins together with the cell debris. Different precipitation pH values (2.5 to 6.0) were investigated. A pH value of 4.2 was selected as a compromise between recovery and improvement of specific activity. A 6 to 8-fold enhancement of the specific activity was obtained, having a r-HBsAg overall yield of about 80%. The influencing presence of a chaotropic salt (potassium thiocyanate) during the acid precipitation step was also studied.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 0749-503X
    Keywords: HARS, Hansenula autonomously replicating sequence ; SUC2, sucrose invertase ; AOX1, alcohol oxidase ; Pollk, polymerase I, fragment klenow ; Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast.The culture conditions for invertase production using a fed-batch culture were studied. More than 1·5×103 U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space. The fermentative process was scaled up to 50 l.Invertase produced from H. polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S. cerevisiae. Using the Western-blot technique, it was observed that invertase secreted from H. polymorpha and invertase secreted from S. cerevisiae showed common antigenic determinants.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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