MURAL - Maynooth University Research Archive Library



    Heterologous expression of human transketolase


    Schenk, Gerhard and Duggleby, Ronald and Nixon, Peter (1998) Heterologous expression of human transketolase. The International Journal of Biochemistry & Cell Biology, 30. pp. 369-378. ISSN 1357-2725

    [img] Download (307kB)


    Share your research

    Twitter Facebook LinkedIn GooglePlus Email more...



    Add this article to your Mendeley library


    Abstract

    Transketolase belongs to the family of thiamin diphosphate dependent enzymes. The aim of this study was to establish a bacterial expression system for human transketolase in order to investigate the functional characteristics of mammalian transketolases. The level of recombinant human enzyme expressed in Escherichia coli was modest. Puri®cation of recombinant transketolase and separation from the E. coli enzyme has been greatly simpli®ed by means of a non-cleavable hexa-histidine tag. The highest speci®c activity was 13.5 U/mg and the Km values were 0.2720.02 and 0.5120.05 mM for the substrates D-xylulose 5-phosphate and D-ribose 5-phosphate, respectively. Binding of cofactors to the apoenzyme showed the expected hysteresis. Without preincubation, the Km values for thiamin diphosphate and for Mg2+ were, respectively, 4.120.8 and 2.520.4 mM, but after 1 h of preincubation these values were 85216 nM and 0.7420.23 mM. The kinetic constants are similar to those of the native enzyme puri®ed from human erythrocytes. Despite the modest expression level the reported system is well suited to a variety of functional studies.

    Item Type: Article
    Keywords: Thiamin diphosphate; Heterologous expression; Protein purifcation; Kinetic parameters; Recombinant human transketolase;
    Academic Unit: Faculty of Science and Engineering > Chemistry
    Item ID: 3687
    Depositing User: Gary Schenk
    Date Deposited: 28 May 2012 14:43
    Journal or Publication Title: The International Journal of Biochemistry & Cell Biology
    Publisher: Elsevier Science Ltd
    Refereed: Yes
    URI:
      Use Licence: This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

      Repository Staff Only(login required)

      View Item Item control page

      Downloads

      Downloads per month over past year

      Origin of downloads