Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli

  • Busso D
  • Delagoutte-Busso B
  • Moras D
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Abstract

We describe here the construction of a 10-Gateway-based vector set applicable for high-throughput cloning and for expressing recombinant proteins in Escherichia coli. Plasmids bear elements required to produce recombinant proteins under control of the T7 promoter and encode different N-terminal partners. Since the vector set is derived from a unique backbone, a consistent comparison of the impact of fusion partner(s) on protein expression and solubility is easily amenable. Finally, a sequence encoding a six-histidine tag has been inserted to be in frame with the cloned open reading frame either at its C terminus or at the N terminus, giving the flexibility of choosing the six-histidine tag location for further purification. To test the applicability of our vector set, expression and solubility profile and six-histidine tag accessibility have been demonstrated for two Bacillus subtilis signaling proteins' encoding genes (SBGP codes E0508 and E0511). © 2005 Elsevier Inc. All rights reserved.

Author-supplied keywords

  • Cloning
  • Expression
  • Fusion
  • Gateway
  • High-throughput screening
  • Recombinant protein
  • Recombinational cloning
  • Structural genomics
  • Tags

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