Expression of synthetic genes encoding fused proteins under tight control of modified regulatory regions of the colicin operon

Abstract

A versatile expression vector system for construction of gene and protein fusions, specific radiolabeling of gene products and high-level protein production is described. Expression cassettes were constructed containing structural genes encoding native and analog forms of connective tissue-activating peptide-III (CTAP-III), β-thromboglobulin, neutrophil-activating protein and modified regulatory sequences derived from the colicin E1 operon. Gene expression was enhanced by changes in the colicin promoter that increased the transcription initiation rate both in vivo and in vitro, and by deletion of a sequence affecting catabolite repression. High-level expression, producing recombinant protein up to 30% of the total cellular protein, was induced rapidly after stimulation of the SOS response by using either mitomycin C or nalidixic acid, by temperature shift using temperature-sensitive mutations in the LexA or RecA proteins, or by UV light. The presence of radiolabeled amino acids during induction resulted in greater than 95% preferential labeling of recombinant proteins. CTAP-III remained stable for more than 6 h following decay of the inducing signal. The use of CTAP-III in protein fusions improved stability of several therapeutically useful proteins including the thrombin-specific inhibitor, hirudin and a cell receptor-binding domain of laminin, when they were produced in Escherichia coli.