Bacterial polypeptide expression employing tryptophan promoter-operator
AssigneeGenentech, Inc.
InventorsDennis G. Kleid, Daniel G. Yansura, Herbert L. Heyneker, Giuseppe F. Miozzari
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Abstract
Methods for the controllable expression in high yields of heterologous proteins under the control of a modified tryptophan promoter-operator system are disclosed. The system employed involves sequential resort to tryptophan repression and derepression, as well as deletion of an attenuator function native to the tryptophan operon. Means are also disclosed for effecting deletions at any given point within a gene fragment and for the ready identification of transformant bacteria via triple ligation procedures.
Claims6
The method of producing a polypeptide product by the expression in bacteria of a structural gene coding therefor which comprises:
- (a) providing a bacterial inoculant transformed with a replicable plasmidic expression vehicle having a sequence of double-stranded DNA comprising, in phase from a first 5′ to a second 3′ end of the coding strand thereof, the elements:
- (i) a bacterial trp promoter-operator system from a bacterial trp operon;
- (ii) nucleotides coding for a ribosome binding site for translation of element (iv);
- (iii) nucleotides coding for a translation start signal for translation of element (iv);
- and
- (iv) a structural gene encoding the amino acid sequence of a heterologous fusion polypeptide comprising 6 amino acids of the trp leader peptide, the at least about the distal third of the trp E polypeptide and a structural gene selected from the group consisting of human growth hormone, human proinsulin, somatostatin, thymosin alpha 1, the A chain of human insulin and the B chain of human insulin;
- said sequence comprising neither any trp attenuation capability nor nucleotides coding for the trp E ribosome binding site;
- b) placing the transformed inoculant in a fermentation vessel and growing the same to a predetermined level in suitable nutrient media containing additive tryptophan sufficient in quantity to repress said promoter-operator system;
- and
- c) depriving said bacteria of said additive while maintaining the viability of said bacteria so as to derepress said system and occasion the expression of the product encoded by said structural gene.
Dependencies are inferred from the claim text.