Tolerance of the Ralstonia eutropha class I polyhydroxyalkanoate synthase for translational fusions to its C terminus reveals a new mode of functional display

Appl Environ Microbiol. 2009 Sep;75(17):5461-6. doi: 10.1128/AEM.01072-09. Epub 2009 Jul 6.

Abstract

Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / genetics*
  • Acyltransferases / metabolism*
  • Artificial Gene Fusion
  • Cupriavidus necator / enzymology*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Periplasmic Binding Proteins / genetics
  • Periplasmic Binding Proteins / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Staphylococcal Protein A / genetics
  • Staphylococcal Protein A / metabolism

Substances

  • Escherichia coli Proteins
  • MalE protein, E coli
  • Periplasmic Binding Proteins
  • Recombinant Fusion Proteins
  • Staphylococcal Protein A
  • Green Fluorescent Proteins
  • Acyltransferases
  • poly(3-hydroxyalkanoic acid) synthase