| 摘要 |
Methods and compositions are provided for regulated expression and secretion of polypeptides in transformed bacterial hosts. A novel class of plasmid cloning vehicles includes a DNA sequence coding for the desired polypeptide (or an insertion site therefor) linked for transcriptional expression in reading phase with four functional fragments derived from the lipoprotein gene of E. coli. The plasmids also include a DNA fragment coding for the signal peptide of the ompA protein of E. coli, positioned such that the desired polypeptide is expressed with the ompA signal peptide at its amino terminus, thereby allowing efficient secretion across the cytoplasmic membrane. The plasmids further include a DNA sequence coding for a specific segment of the E. coli lac promoter-operator, which is positioned in the proper orientation for transcriptional expression of the desired polypeptide, as well as a separate functional E. coli lacI gene coding for the associated repressor molecule which can interact with the lac promoter-operator to prevent transcription therefrom. Expression of the desired polypeptide is under the control of both the lipoprotein promoter and the lac promoter-operator, although transcription from either promoter is normally blocked by the repressor molecule. However, the repressor can be selectively inactivated by means of an inducer molecule to permit transcriptional expression of the desired polypeptide from both promoters. The methods utilize such plasmids to introduce genetic capability into micro-organisms for the production of normally secreted proteins, such as medically or commercially useful hormones, enzymes, immunogenic proteins, or intermediates therefor, but only in the presence of an appropriate inducer.
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