Purification and characterization of the Thermus thermophilus HB8 RecX protein.
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 alpha-helical content of ttRecX is 54% and that it is stable up to 80 degrees 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.
Jimbo
Koutaro
K
International Graduate School of Arts and Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Inoue
Jin
J
Masuda
Tokiha
T
Shibata
Takehiko
T
Mikawa
Tsutomu
T
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D017433:Protein Structure, Secondary; D011938:Rec A Recombinases / Q000037:antagonists & inhibitors; D016963:Thermus thermophilus / Q000737:chemistry*
D001426:Bacterial Proteins; C430523:RecX protein, Xanthomonas campestris; D011938:Rec A Recombinases