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

Anal Biochem. 2005 Aug 15;343(2):313-21. doi: 10.1016/j.ab.2005.05.015.


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).

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Bacillus subtilis / genetics
  • Cloning, Molecular / methods*
  • Electrophoresis, Polyacrylamide Gel / methods*
  • Escherichia coli / genetics*
  • Genetic Vectors / genetics*
  • Histidine / chemistry
  • Histidine / genetics
  • Recombinant Proteins / genetics


  • Recombinant Proteins
  • Histidine