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

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 con served 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 al l the conserved motifs. We constructed a truncated ttRecJ gene that corresp onds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60 degreesC. Siz e exclusion chromatography indicated that ttRecJ exists in several oligomer ic states, whereas cd-ttRecJ is monomeric in solution Both proteins have 5' -->3' exonuclease activity, which was enhanced by increasing the temperatur e to 50 degreesC. Mg2+, Mn2+ or CO2+ ions were required to activate both pr oteins, whereas Ca2+ and Zn2+ had no effects.