Amplification and purification of exonuclease I from Escherichia coli K12

Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been develope...

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Bibliographic Details
Published in:The Journal of biological chemistry 1983-05, Vol.258 (10), p.6340-6343
Main Authors: Prasher, D C, Conarro, L, Kushner, S R
Format: Article
Language:English
Subjects:
DNA - metabolism
Escherichia coli - enzymology
Escherichia coli - genetics
Exodeoxyribonucleases - genetics
Exodeoxyribonucleases - isolation & purification
Molecular Weight
Plasmids
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Summary:Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities. The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M. (1974) J. Biol. Chem. 249, 5379-5381). Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)32414-1