Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli.
Fiche publication
Date publication
août 2005
Auteurs
Membres identifiés du Cancéropôle Est :
Dr MORAS Dino
Tous les auteurs :
Busso D, Delagoutte-Busso B, Moras D
Lien Pubmed
Résumé
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).
Référence
Anal Biochem. 2005 Aug 15;343(2):313-21.