Ruiz-Masó José A, López-Zumel Consuelo, Menéndez Margarita, Espinosa Manuel, del Solar Gloria
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Velázquez, 144, E-28006 Madrid, Spain.
Biochim Biophys Acta. 2004 Jan 14;1696(1):113-9. doi: 10.1016/j.bbapap.2003.09.010.
The promiscuous rolling circle (RC)-replicating plasmid pMV158 encodes the 210-amino-acid initiator of replication protein, RepB. The protein relaxes supercoiled cognate DNA in a topoisomeraseI-like manner. A new vector and procedure for overproduction, scaling-up, and purification of the protein has been developed. RepB purified as a hexamer in solution, as shown by analytical ultracentrifugation assays. Circular dichroism (CD) of RepB indicated that the protein has an estimated content of around 33% alpha-helices and 20% beta-strands. Characterisation of temperature-induced transitions of the protein showed an irreversible change in the spectra when the temperature was raised above 35 degrees C, indicating that the protein undergoes a conformational change that could account for the relatively high optimal temperature of the RepB-mediated cleavage.
杂乱滚环（RC）复制质粒pMV158编码210个氨基酸的复制起始蛋白RepB。该蛋白以拓扑异构酶I样方式松弛超螺旋同源DNA。已开发出一种用于该蛋白过量生产、放大和纯化的新载体及方法。如分析超速离心测定所示，纯化的RepB在溶液中以六聚体形式存在。RepB的圆二色性（CD）表明，该蛋白估计含有约33%的α-螺旋和20%的β-链。对该蛋白温度诱导转变的表征显示，当温度升至35摄氏度以上时，光谱发生不可逆变化，表明该蛋白发生了构象变化，这可能解释了RepB介导切割的相对较高的最适温度。
