Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1985-12-05
1990-04-03
Teskin, Robin
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 435320, 435 91, 435 21, 435 34, 435 6, 435818, 435849, 435196, 536 27, 935 47, 935 48, 935 72, C12P 2100, C12P 2102, C12N 1200
Patent
active
049140256
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.
REFERENCES:
Casadaban, M. J. et al., .beta.-Galactosidase Gene Fusions for Analyzing Gene Expression in E. coli and Yeast; Methods in Enzymology (1983), vol. 100, pp. 293-308.
Stachel, S. E. et al.; A Tn3 lacZ Transposon for the Random Generation of .beta.-galactosidase Gene Fusions: Application to the Analysis of Gene Expression in Agrobacterium; EMBO Journal, 4 (4), pp. 891-898 (1985).
Berg, D. E. and C. M. Berg; The Prokaryotic Transposable Element Tn5; Biotechnology 1: 417-435 (1983).
Manoil, C. and J. Beckwith; TNphoA: A Transposon Probe for Protein Export Signals; Proc. Nat'l. Acad. Sci., 82, pp. 8129-8133 (1985).
Guo L-H. and R. Wu; Exonuclease III: Use for DNA Sequence Analysis and in Specific Deletions of Nucleotides; Methods in Enzymology, 100 (1983), pp. 60-95.
Hoffman, C. S. and A. Wright; Fusions of Secreted Proteins to Alkaline Phosphatase: An Approach for Studying Protien Secretion; Proc. Nat'l. Acad. Sci., 82 (1985), pp. 5107-5111.
Kroos, L. and D. Kaiser; Construction of Tn5 lac, a Transposon That Fuses lacz Expression to Exogenous Promoters, and Its Introduction into M. xanthus; Proc. Nat'l. Acad. Sci., 81 (1984), pp. 5816-5820.
Michaels and Beckwith, Annu. Rev. Microbiol., vol. 36, pp. 435-465.
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Beckwith Jonathan
Hoffman Charles S.
Isberg Ralph R.
Manoil Colin
Syvanen Michael
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