Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli.
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).
Département de Biologie et de Génomique Structurales, IGBMC, CNRS/INSERM/Université Louis Pasteur, Parc d'Innovation, 1 rue Laurent Fries, BP10142, 67404 Illkirch Cedex, France. djbusso@igbmc.u-strasbg.fr
Didier
D
Busso
Bénédicte
B
Delagoutte-Busso
Dino
D
Moras
D001412:Bacillus subtilis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
D011994:Recombinant Proteins; D006639:Histidine