Generation of an AraC-araBAD promoter-regulated T7 expression system.
An alternative and facile delivery system for T7 RNA polymerase has been devised and constructed. T7 gene 1 has been placed under control of the araBAD promoter element regulated by the AraC protein. Cotransformation of the resultant plasmid, pTara, with one containing a target gene under T7 promoter-regulated expression potentially allows repression by glucose and induction by arabinose in the range of 0.5 to 20 mM sugar concentration. To demonstrate the efficacy of this expression system, the p53 gene under T7 promoter control in two different plasmids was expressed in Escherichia coli using pTara as the source of T7 RNA polymerase. Repression and induction of p53 were achieved in both a lower and higher copy number plasmid, although the levels of induction were higher with the lower copy number expression vector. Cotransformation of an expression plasmid with pTara provides a low-cost method of T7 RNA polymerase-regulated expression that can be fine-tuned using glucose and arabinose concentrations to balance protein expression with potential solubility or toxicity problems.
Wycuff
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DR
Department of Biochemistry, Rice University, Houston, Texas, 77251-1892, USA.
Matthews
K S
KS
D001089:Arabinose / Q000494:pharmacology; D017123:Bacteriophage T7 / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D016158:Genes, p53; D005947:Glucose / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D014764:Viral Proteins
D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D001089:Arabinose; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D005947:Glucose