Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli.
A generic vector, pASK75, was developed for the synthesis of foreign proteins in Escherichia coli under transcriptional control of the tetA promoter/operator. Tight regulation was achieved by placing the structural gene for the tet repressor, as a transcriptional fusion, downstream from the beta-lactamase-encoding gene (bla) on the same plasmid. Strong expression of the foreign gene was conveniently induced by adding anhydrotetracycline at a low concentration. Using the production of a recombinant murine immunoglobulin F(ab) fragment as an example, the system was shown to function independently of the host-strain background and to be extremely well repressed in the absence of the inducer. Thus, it represents an economic and independent alternative to IPTG-inducible promoter constructs. Additional features of pASK75 include a signal sequence and a multiple cloning site followed by a region encoding the Strep tag affinity peptide to facilitate purification of a bacterially produced protein.
Skerra
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Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
D000595:Amino Acid Sequence; D000818:Animals; D017920:Antiporters / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics; D013291:Streptococcus / Q000235:genetics; D013752:Tetracycline / Q000096:biosynthesis*; D001618:beta-Lactamases / Q000235:genetics
D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D012097:Repressor Proteins; C079051:tetA protein, Bacteria; C034811:tetracycline resistance-encoding transposon repressor protein; D001618:beta-Lactamases; D013752:Tetracycline