Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1984-08-07
1988-08-30
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
435 68, 435 70, 4351723, 435320, 435253, 435255, 935 14, 935 28, 935 29, 935 41, 935 43, C12N 120, C12N 500, C12N 912, C12N 2100, C12N 2102, C12N 1500
Patent
active
047677082
ABSTRACT:
Restriction enzymes are used to remove from DNA a complete and undamaged structural gene coding region for the expression of DNA polymerase I (polA) without the gene's natural promoter or with only a significantly damaged portion of the gene's natural promoter. Also by the use of restriction enzymes, a segment from a plasmid cloning vector is excised at a position adjacent to a promoter which is conditionally controllable and may be more powerful than the damaged or removed promoter. The gene for DNA polymerase I is enzymatically cloned into said vector at the position of said removed segment and adjacent to said conditionally controllable promoter. Multicopies of the cloned vector are introduced into a host baterial strain (E. coli). The host strain is then cultured so that the cell colony grows and replicates new generations containing replicated foreign plasmid. During such said replication the activity of said controllable promoter is repressed. After the cell colony has grown, the repression of said controllable promoter is removed and the cells express an amplified amount of DNA polymerase I which is lethal or inhibitory to the cells. An improved procedure is disclosed comprising a sequence of steps for harvesting purified DNA polymerase I.
REFERENCES:
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Loeb et al. In. The Enzymes, 3rd Ed., Boyer, ed. Academic Press, Inc. 1974. pp. 173-188.
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"Construction of a Plasmid That Overproduces the Large Proteolytic Fragment (Klenow fragment) of DNA Polymerase I of Escherichia coli", C. M. Joyce and N. D. F. Grindley (1983) Proc. Natl. Acad. Sci., U.S.A. 80, pp. 1830-1834.
"Plasmid Cloning and Expression of the E. coli polA+ gene in S. Cerevisiae", A. Spanos and S. G. Sedgwick, Current Genetics (1984), 8, pp. 333-340 (exact publication date not given on article).
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Kornberg, In DNA Replication, pp. 169, 172-181, 187, 201-205, W. H. Freeman and Co., 1980.
Brown William E.
Minkley, Jr. Edwin G.
Carnegie Mellon University
Cornelius Andrew J.
Kass Alan P.
Mays Thomas D.
Wiseman Thomas G.
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