Enzyme amplification and purification process for producing DNA

Details Enzyme amplification and purification process for producing DNA Enzyme amplification and purification process for producing DNA

1995-03-30

2000-01-25

Achutamurthy, Ponnathapura

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

4352523, 43525233, 4353201, 536 231, 536 232, C12N 912, C12N 1570

Patent

active

060177454

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.

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