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

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).
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https://hal.archives-ouvertes.fr/hal-00187474
Contributor : Jean-Luc Toussaint <>
Submitted on : Wednesday, November 14, 2007 - 4:02:40 PM
Last modification on : Thursday, April 23, 2020 - 2:26:26 PM

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Didier Busso, Bénédicte Delagoutte-Busso, Dino Moras. Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli.. Analytical Biochemistry, Elsevier Masson, 2005, 343 (2), pp.313-21. ⟨10.1016/j.ab.2005.05.015⟩. ⟨hal-00187474⟩

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