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

Didier Busso (1) , Bénédicte Delagoutte-Busso (1) , Dino Moras (1)

1 IGBMC - Institut de génétique et biologie moléculaire et cellulaire

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).

Domaines: Biologie moléculaire

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, 2005, 343 (2), pp.313-21. ⟨10.1016/j.ab.2005.05.015⟩. ⟨hal-00187474⟩