公开(公告)号：US4914025A

公开(公告)日：1990-04-03

申请号：US805486

申请日：1985-12-05

Applicant: Colin Manoil ,  Jonathan Beckwith ,  Michael Syvanen ,  Ralph R. Isberg ,  Charles S. Hoffman ,  Andrew Wright

Inventor： Colin Manoil ,  Jonathan Beckwith ,  Michael Syvanen ,  Ralph R. Isberg ,  Charles S. Hoffman ,  Andrew Wright

IPC: C12N15/10 ,  C12N15/62

CPC classification number: C12N15/10 ,  C12N15/625 ,  C07K2319/02 ,  C07K2319/61 ,  Y10S435/848 ,  Y10S435/849

Abstract: An export sequence of export DNA can be identified by transforming a population of cells with a vector having a transposon that includes a structural gene encoding a detectable compound, positioned between insertion sequences. The structural gene encodes a detectable compound that, in wild-type organisms, is translated with an export peptide effective to export the compound. Since the transposon lacks DNA coding for an export sequence capable of exporting the detectable gene product, transformants that export the gene product include a DNA fusion of the transposon to export DNA from the parent cell, in a position to allow expression of the fused DNA. The transformants are analyzed to locate the export DNA or a gene comprising it, and the position of the export DNA in the cell's genome is determined; the orientation of the insertion also is determined. Having identified cell genes that naturally contain export DNA, the export DNA is cloned and fused to a gene encoding a product whose production and export are desired. For example, a cell is engineered to produce and export a desired product, to enable easier substance recovery and to improve cell tolerance for high levels of the desired product.

Abstract translation: 可以通过用含有位于插入序列之间的包含编码可检测化合物的结构基因的转座子转化细胞群来鉴定出口DNA的出口序列。 结构基因编码可检测的化合物，其在野生型生物体中用有效导出化合物的出口肽翻译。 由于转座子缺乏编码能够输出可检测基因产物的出口序列的DNA，所以导出基因产物的转化子包括转座子的DNA融合，以从母细胞输出DNA，以允许表达融合的DNA。 分析转化体以定位出口DNA或包含其的基因，确定出口DNA在细胞基因组中的位置; 插入的方向也被确定。 鉴定出天然含有出口DNA的细胞基因后，将出口DNA克隆并融合到编码其生产和出口的产品的基因上。 例如，将细胞工程化以产生和输出所需产物，以使更容易的物质回收和改善对高水平所需产物的细胞耐受性。