A versatile and highly repressible Escherichia coli expression system based on invertible promoters: expression of a gene encoding a toxic product.
A very flexible and tightly regulatable expression system has been constructed. It uses the principle of invertible promoters [Podhajska et al., Gene 40 (1985) 163-168]. Here, we describe the construction of a plasmid that provides the integrase, which causes promoter inversion in a tightly regulated fashion, as well as modified plasmids carrying the invertible module. The way the integrase is provided on a separate plasmid closely mimicks expression of the integrase from a lambda lysogen. Thus, the flexibility of the original system is considerably extended by making it strain-independent without compromising the tight regulation. We present the expression of a single-chain T-cell receptor fragment as an example of application, in order to illustrate the properties of this expression system.
Wülfing
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Protein Engineering Group, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Plückthun
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D019426:Integrases; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011948:Receptors, Antigen, T-Cell / Q000235:genetics
D011948:Receptors, Antigen, T-Cell; D004254:DNA Nucleotidyltransferases; D019426:Integrases