Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain

Atsushi Yamagata, Ryoji Masui, Yoshimitsu Kakuta, Seiki Kuramitsu, Keiichi Fukuyama

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

A recJ homolog was cloned from the extremely thermophilic bacterium Thermus themophilus HB8. It encodes a 527 amino acid protein that has 33% identity to Escherichia coliRecJ protein and includes the characteristic motifs conserved among RecJ homologs. Although T.thermophilus RecJ protein (ttRecJ) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. Limited proteolysis showed that ttRecJ has a protease-resistant core domain, which includes all the conserved motifs. We constructed a truncated ttRecJ gene that corresponds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60°C. Size exclusion chromatography indicated that ttRecJ exists in several oligomeric states, whereas cd-ttRecJ is monomeric in solution. Both proteins have 5′→3′ exonuclease activity, which was enhanced by increasing the temperature to 50°C. Mg2+, Mn2+ or Co2+ ions were required to activate both proteins, whereas Ca2+ and Zn2+ had no effects.

Original languageEnglish
Pages (from-to)4617-4624
Number of pages8
JournalNucleic Acids Research
Volume29
Issue number22
Publication statusPublished - Nov 15 2001
Externally publishedYes

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Thermus thermophilus
Proteins
Thermus
Escherichia
Exonucleases
Inclusion Bodies
Proteolysis
Gel Chromatography

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain. / Yamagata, Atsushi; Masui, Ryoji; Kakuta, Yoshimitsu; Kuramitsu, Seiki; Fukuyama, Keiichi.

In: Nucleic Acids Research, Vol. 29, No. 22, 15.11.2001, p. 4617-4624.

Research output: Contribution to journalArticle

Yamagata, Atsushi ; Masui, Ryoji ; Kakuta, Yoshimitsu ; Kuramitsu, Seiki ; Fukuyama, Keiichi. / Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain. In: Nucleic Acids Research. 2001 ; Vol. 29, No. 22. pp. 4617-4624.
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