Journal
ANALYTICAL BIOCHEMISTRY
Volume 343, Issue 2, Pages 313-321Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.05.015
Keywords
cloning; expression; fusion; gateway; high-throughput screening; recombinant protein; recombinational cloning; structural genomics; tags
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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). (C) 2005 Elsevier Inc. All rights reserved.
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