Construction of an E. coli strain overproducing the Tn10-encoded TET repressor and its use for large scale purification

Abstract

Overproduction of the repressor protein from the Tn10-encoded tetracycline resistance operon is achieved by placement of the respective gene under control of bacteriophage lambda promoter PL in a vector-host system. All cloning steps have to be carried out under repressed conditions to assure survival of the cell. The cI 857 mutation is used to control expression of the tetR gene in large scale fermentation. After induction, the overproducing Escherichia coli strain continues to grow for 2.5 generations before growth terminates. In the expression phase, active TET repressor comprises up to 13% of the total soluble protein. A procedure is described to purify the TET repressor protein to homogeneity on a large scale. Starting from a 10 litre culture, approximately 250 mg of homogeneous, active TET repressor are obtained. The amino acid sequence of the N and C termini are in agreement with the gene start and stop determined from the nucleotide sequence of the Tn10 tetR gene.