Purification and characterization of the Thermus thermophilus HB8 RecX protein

Koutaro Jimbo; Jin Inoue; Tokiha Masuda; Takehiko Shibata; Tsutomu Mikawa

doi:10.1016/j.pep.2006.09.001

Purification and characterization of the Thermus thermophilus HB8 RecX protein

Koutaro Jimbo, Jin Inoue, Tokiha Masuda, Takehiko Shibata, Tsutomu Mikawa

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

3 Citations (Scopus)

Abstract

The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the α-helical content of ttRecX is 54% and that it is stable up to 80 °C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.

Original language	English
Pages (from-to)	320-323
Number of pages	4
Journal	Protein Expression and Purification
Volume	51
Issue number	2
DOIs	https://doi.org/10.1016/j.pep.2006.09.001
Publication status	Published - 2007 Feb
Externally published	Yes

Keywords

RecA
Recombination
Thermostable
Thermus thermophilus

ASJC Scopus subject areas

Biotechnology

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10.1016/j.pep.2006.09.001

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title = "Purification and characterization of the Thermus thermophilus HB8 RecX protein",

abstract = "The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the α-helical content of ttRecX is 54% and that it is stable up to 80 °C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.",

keywords = "RecA, Recombination, Thermostable, Thermus thermophilus",

author = "Koutaro Jimbo and Jin Inoue and Tokiha Masuda and Takehiko Shibata and Tsutomu Mikawa",

note = "Funding Information: We thank Dr. Seiki Kuramitsu for advice on the T. thermophilus genes used in this study. This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science to T.S. and T.M.",

year = "2007",

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doi = "10.1016/j.pep.2006.09.001",

language = "English",

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AU - Jimbo, Koutaro

AU - Inoue, Jin

AU - Masuda, Tokiha

AU - Shibata, Takehiko

AU - Mikawa, Tsutomu

N1 - Funding Information: We thank Dr. Seiki Kuramitsu for advice on the T. thermophilus genes used in this study. This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science to T.S. and T.M.

PY - 2007/2

Y1 - 2007/2

N2 - The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the α-helical content of ttRecX is 54% and that it is stable up to 80 °C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.

AB - The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the α-helical content of ttRecX is 54% and that it is stable up to 80 °C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.

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KW - Recombination

KW - Thermostable

KW - Thermus thermophilus

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Purification and characterization of the Thermus thermophilus HB8 RecX protein

Abstract

Keywords

ASJC Scopus subject areas

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