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Nucleic Acids Research, 2001, Vol. 29, No. 22 4617-4624
© 2001 Oxford University Press

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

Atsushi Yamagata1, Ryoji Masui1,2, Yoshimitsu Kakuta1,2, Seiki Kuramitsu1,2 and Keiichi Fukuyama1,2,*

1Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan and 2RIKEN Harima Institute/SPring-8, 1-1-1 Koto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan

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 coli RecJ 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.

* To whom correspondence should be addressed at: 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan. Tel: +81 6 6850 5422; Fax: +81 6 6850 5425; Email: fukuyama{at}bio.sci.osaka-u.ac.jp


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Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Yamagata, Y. Kakuta, R. Masui, and K. Fukuyama
The crystal structure of exonuclease RecJ bound to Mn2+ ion suggests how its characteristic motifs are involved in exonuclease activity
PNAS, April 30, 2002; 99(9): 5908 - 5912.
[Abstract] [Full Text] [PDF]


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