Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits

Paul E. Boehmer; Peter T. Emmerson

doi:10.1016/0378-1119(91)90529-K

Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits

Paul E. Boehmer, Peter T. Emmerson

Basic Medical Sciences, Phoenix

Research output: Contribution to journal › Article › peer-review

39 Scopus citations

Abstract

The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3. The overproduced proteins occur at levels amounting to approx. 10% of total cellular protein. Strains harbouring these overexpression plasmids have been used to purify the RecB, RecC and RecD protein subunits, as well as the RecBCD holoenzyme. The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme.

Original language	English (US)
Pages (from-to)	1-6
Number of pages	6
Journal	Gene
Volume	102
Issue number	1
DOIs	https://doi.org/10.1016/0378-1119(91)90529-K
State	Published - Jun 15 1991

Keywords

Recombinant proteins
functional enzyme
overexpression
purification

ASJC Scopus subject areas

Genetics

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10.1016/0378-1119(91)90529-K

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title = "Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits",

abstract = "The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3. The overproduced proteins occur at levels amounting to approx. 10% of total cellular protein. Strains harbouring these overexpression plasmids have been used to purify the RecB, RecC and RecD protein subunits, as well as the RecBCD holoenzyme. The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme.",

keywords = "Recombinant proteins, functional enzyme, overexpression, purification",

author = "Boehmer, {Paul E.} and Emmerson, {Peter T.}",

note = "Funding Information: We thank June Ramsay for technical assistance. This work was supported by a grant from the Medical Research Council. P.E.B. was the recipient of a Cookson Industrial Materials Studentship from the University of Newcastle upon Tyne.",

year = "1991",

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doi = "10.1016/0378-1119(91)90529-K",

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T1 - Escherichia coli RecBCD enzyme

T2 - inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits

AU - Boehmer, Paul E.

AU - Emmerson, Peter T.

N1 - Funding Information: We thank June Ramsay for technical assistance. This work was supported by a grant from the Medical Research Council. P.E.B. was the recipient of a Cookson Industrial Materials Studentship from the University of Newcastle upon Tyne.

PY - 1991/6/15

Y1 - 1991/6/15

N2 - The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3. The overproduced proteins occur at levels amounting to approx. 10% of total cellular protein. Strains harbouring these overexpression plasmids have been used to purify the RecB, RecC and RecD protein subunits, as well as the RecBCD holoenzyme. The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme.

AB - The intracellular levels of the Escherichia coli RecBCD proteins have been amplified by fusing the recBCD genes to the strong tac promoter/operator in the expression vector, pKK223-3. The overproduced proteins occur at levels amounting to approx. 10% of total cellular protein. Strains harbouring these overexpression plasmids have been used to purify the RecB, RecC and RecD protein subunits, as well as the RecBCD holoenzyme. The individually purified protein subunits can be used to reconstitute the ATP-dependent DNase activity of the RecBCD enzyme.

KW - Recombinant proteins

KW - functional enzyme

KW - overexpression

KW - purification

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Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits

Abstract

Keywords

ASJC Scopus subject areas

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