Overproduction and characterization of the uracil-DNA glycosylase inhibitor of bacteriophage PBS2

Wang Zhigang; Debra G. Smith; Dale W. Mosbaugh

doi:10.1016/0378-1119(91)90030-F

Overproduction and characterization of the uracil-DNA glycosylase inhibitor of bacteriophage PBS2

Wang Zhigang, Debra G. Smith, Dale W. Mosbaugh

Toxicology and Cancer Biology

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

44 Scopus citations

Abstract

A plasmid expression vector (pZWtacl) was constructed which allowed inducible overexpression of the uracil-DNA glycosylase (Ung) inhibitor (Ugi)-encoding gene (ugi) in Escherichia coli. In this plasmid, the ugi gene was under the control of both its own promoter and the tac promoter. Constitutive expression of the ugi was observed in the absence of isopropyl-β-d-thiogalactopyranoside (IPTG). In the presence of 1 mM IPTG, the Ugi protein was overproduced to an approx. 16-fold higher level, and accounted for approx. 19% of the total soluble cellular proteins. Following high-level production in E. coli, the Ugi protein was purified to apparent homogeneity. Using E. coli Ung, we observed that Ugi inactivated the enzyme in a noncompetitive manner. Kinetic studies revealed a K_i value (0.14 μM) of approx. twelve-fold lower than K_m value (1.7 μM) of glycosylase. Ugi did not act synergistically with free uracil to inhibit E. coli Ung suggesting that uracil and Ugi could share a similar mode of inhibition.

Original language	English
Pages (from-to)	31-37
Number of pages	7
Journal	Gene
Volume	99
Issue number	1
DOIs	https://doi.org/10.1016/0378-1119(91)90030-F
State	Published - Mar 1 1991

Bibliographical note

Funding Information:
t We thank Xiaohua Wu for her excellentt echnical assistancea nd MatthewL ongleyf or criticale valuationo f the manuscriptW. e alsot hankD r. Bruce Duncanf or pro-vidingt hec lonedE . coli uracil-DNA glycosylaseT.h is work was supportedb y National Instituteso f Health Grant I GM32823a ndt hef ormerC laytonF oundationB iochemical + 9437f rom the OregonA griculturalE xperimentS tation. Institutea t the Universityo f Texas.T echnicalr eportN o.

Funding

t We thank Xiaohua Wu for her excellentt echnical assistancea nd MatthewL ongleyf or criticale valuationo f the manuscriptW. e alsot hankD r. Bruce Duncanf or pro-vidingt hec lonedE . coli uracil-DNA glycosylaseT.h is work was supportedb y National Instituteso f Health Grant I GM32823a ndt hef ormerC laytonF oundationB iochemical + 9437f rom the OregonA griculturalE xperimentS tation. Institutea t the Universityo f Texas.T echnicalr eportN o.

Funders	Funder number
National Instituteso f Health	I GM32823a
OregonA griculturalE
National Institute of General Medical Sciences	R01GM032823

Keywords

Bacillus subtilis phage
DNA repair
Recombinant DNA
overexpression vector
tac promoter
uracil-DNA glycosylase

ASJC Scopus subject areas

Genetics

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10.1016/0378-1119(91)90030-F

Cite this

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title = "Overproduction and characterization of the uracil-DNA glycosylase inhibitor of bacteriophage PBS2",

abstract = "A plasmid expression vector (pZWtacl) was constructed which allowed inducible overexpression of the uracil-DNA glycosylase (Ung) inhibitor (Ugi)-encoding gene (ugi) in Escherichia coli. In this plasmid, the ugi gene was under the control of both its own promoter and the tac promoter. Constitutive expression of the ugi was observed in the absence of isopropyl-β-d-thiogalactopyranoside (IPTG). In the presence of 1 mM IPTG, the Ugi protein was overproduced to an approx. 16-fold higher level, and accounted for approx. 19\% of the total soluble cellular proteins. Following high-level production in E. coli, the Ugi protein was purified to apparent homogeneity. Using E. coli Ung, we observed that Ugi inactivated the enzyme in a noncompetitive manner. Kinetic studies revealed a Ki value (0.14 μM) of approx. twelve-fold lower than Km value (1.7 μM) of glycosylase. Ugi did not act synergistically with free uracil to inhibit E. coli Ung suggesting that uracil and Ugi could share a similar mode of inhibition.",

keywords = "Bacillus subtilis phage, DNA repair, Recombinant DNA, overexpression vector, tac promoter, uracil-DNA glycosylase",

author = "Wang Zhigang and Smith, \{Debra G.\} and Mosbaugh, \{Dale W.\}",

note = "Funding Information: t We thank Xiaohua Wu for her excellentt echnical assistancea nd MatthewL ongleyf or criticale valuationo f the manuscriptW. e alsot hankD r. Bruce Duncanf or pro-vidingt hec lonedE . coli uracil-DNA glycosylaseT.h is work was supportedb y National Instituteso f Health Grant I GM32823a ndt hef ormerC laytonF oundationB iochemical + 9437f rom the OregonA griculturalE xperimentS tation. Institutea t the Universityo f Texas.T echnicalr eportN o.",

year = "1991",

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language = "English",

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AU - Zhigang, Wang

AU - Smith, Debra G.

AU - Mosbaugh, Dale W.

N1 - Funding Information: t We thank Xiaohua Wu for her excellentt echnical assistancea nd MatthewL ongleyf or criticale valuationo f the manuscriptW. e alsot hankD r. Bruce Duncanf or pro-vidingt hec lonedE . coli uracil-DNA glycosylaseT.h is work was supportedb y National Instituteso f Health Grant I GM32823a ndt hef ormerC laytonF oundationB iochemical + 9437f rom the OregonA griculturalE xperimentS tation. Institutea t the Universityo f Texas.T echnicalr eportN o.

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Y1 - 1991/3/1

N2 - A plasmid expression vector (pZWtacl) was constructed which allowed inducible overexpression of the uracil-DNA glycosylase (Ung) inhibitor (Ugi)-encoding gene (ugi) in Escherichia coli. In this plasmid, the ugi gene was under the control of both its own promoter and the tac promoter. Constitutive expression of the ugi was observed in the absence of isopropyl-β-d-thiogalactopyranoside (IPTG). In the presence of 1 mM IPTG, the Ugi protein was overproduced to an approx. 16-fold higher level, and accounted for approx. 19% of the total soluble cellular proteins. Following high-level production in E. coli, the Ugi protein was purified to apparent homogeneity. Using E. coli Ung, we observed that Ugi inactivated the enzyme in a noncompetitive manner. Kinetic studies revealed a Ki value (0.14 μM) of approx. twelve-fold lower than Km value (1.7 μM) of glycosylase. Ugi did not act synergistically with free uracil to inhibit E. coli Ung suggesting that uracil and Ugi could share a similar mode of inhibition.

AB - A plasmid expression vector (pZWtacl) was constructed which allowed inducible overexpression of the uracil-DNA glycosylase (Ung) inhibitor (Ugi)-encoding gene (ugi) in Escherichia coli. In this plasmid, the ugi gene was under the control of both its own promoter and the tac promoter. Constitutive expression of the ugi was observed in the absence of isopropyl-β-d-thiogalactopyranoside (IPTG). In the presence of 1 mM IPTG, the Ugi protein was overproduced to an approx. 16-fold higher level, and accounted for approx. 19% of the total soluble cellular proteins. Following high-level production in E. coli, the Ugi protein was purified to apparent homogeneity. Using E. coli Ung, we observed that Ugi inactivated the enzyme in a noncompetitive manner. Kinetic studies revealed a Ki value (0.14 μM) of approx. twelve-fold lower than Km value (1.7 μM) of glycosylase. Ugi did not act synergistically with free uracil to inhibit E. coli Ung suggesting that uracil and Ugi could share a similar mode of inhibition.

KW - Bacillus subtilis phage

KW - DNA repair

KW - Recombinant DNA

KW - overexpression vector

KW - tac promoter

KW - uracil-DNA glycosylase

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JO - Gene

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Overproduction and characterization of the uracil-DNA glycosylase inhibitor of bacteriophage PBS2

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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Cite this