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  • Biochemistry and Biotechnology  (3)
  • 1
    Electronic Resource
    Electronic Resource
    Expression of epoxide hydrolase in insect cells: A focus on the infected cell (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 42 (1993), S. 240-246 
    Details
    ISSN: 0006-3592
    Keywords: insect cell culture ; baculovirus expression ; serum-free media ; insect cell metabolism ; cell phase ; high cell density expression ; epoxide hydrolase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Insect cell culture and the baculovirus vector expression system have emerged to be a promising production technique for heterologous proteins. In this article, expression characteristics for membrane-bound epoxide hydrolase are examined. A generic process is presented whereby cells are grown in serum-free media supplemented with serum and then resuspended in serum-free media to simplify purification after infection. The infected cells retain significant metabolic activity during the postinfection stage. Thus, maintaining nutrient supply during the postinfection period is critical, and a low stirring rate will result in oxygen depletion and shift the metabolism of the infected cells toward lactate production which then lowers product yield. This is the first report indicating that glucose is supplied from sucrose decomposition and then metabolized for viral DNA and recombinant protein production in recombinant baculovirus insect expression system. © 1993 John Wiley & Sons, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260420212
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  • 2
    Electronic Resource
    Electronic Resource
    Purification of a recombinant protein produced in a baculovirus expression system by immobilized metal affinity chromatography (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 43 (1994), S. 349-356 
    Details
    ISSN: 0006-3592
    Keywords: immobilized metal ion affinity chmotagraphy ; baculovirus expression system ; infectious bursal disease virus ; protein purification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Over the past 10 years, the baculovirus-insect cell system has become a powerful and versatile tool for the expression of a variety of heterologous proteins. In order to simplify separation of a cloned protein from the baculovirus-insect expression system, we have cloned a gene encoding for the protein of interest, a structural protein (VP2) of a strain (E/DEL) of infectious bursal disease virus (IBDV), with a metal ion binding site (His)5 at its C-terminus. This chimeric protein (VP2H) has been expressed and one-step affinity purified with immobilized metal ions (Ni+2). With antigen capture-enzyme-linked immunosorbent assay (AC-ELISA), we determined that the conformation of this chimeric protein was no different from the recombinant wild-type VP2 protein. However, the two proteins (VP2 and VP2H) can be distinguished and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected immunologically following Western blotting. © 1994 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260430502
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Pulsed field separation of large supercoiled and open-circular DNAs and its application to bacterial artificial chromosome cloning (1995)
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 1-7 
    Details
    ISSN: 0173-0835
    Keywords: Cloning ; Bacterial artificial chromosome ; Supercoiled DNA ; Open-circular DNA ; Pulsed field gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We have studied the separation of large (80-300 kbp) supercoiled (SC) DNA in conventional agarose gel electrophoresis, field inversion gel electrophoresis (FIGE) and pulsed field gel electrophoresis (PFGE). DNA migration was measured under a variety of electrophoretic conditions including different switch times, temperatures, agarose concentrations, and voltage gradients. The migration of SC DNA was found to be inversely proportional to its molecular weight in the three electrophoresis systems tested. In conventional agarose electrophoresis, voltage gradient was found to be the determining parameter in the separation of SC DNA. Unlike large linear DNAs, the migration of SC DNA was found to be independent of switch time in PFGE and FIGE. Broad DNA bands were observed in prolonged FIGE runs. In addition, we have also studied the migration of open-circular (OC) DNA (80 and 100 kbp) in pulsed field gel electrophoresis. Eighty kbp OC DNA can migrate into agarose gels under certain pulsed field conditions whereas 100 kbp OC DNA was trapped at the wells. Based on electrophoretic conditions described in this report, we can determine the size of bacterial artificial chromosome (BAC) clones without restriction enzyme digestion and have enriched the percentage of larger size clones in BAC cloning.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150160102
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    Paper (German National Licenses)
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