Ueki Nobuhiko, Ochiai Yoshihiro
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Japan.
Biosci Biotechnol Biochem. 2005 Oct;69(10):1935-43. doi: 10.1271/bbb.69.1935.
An expression system of recombinant myoglobins (Mb) of 3 scombridae fish species was constructed. The stability of these Mbs was compared with native Mbs purified from slow skeletal muscle. The addition of hemin during the cultivation of an Escherichia coli strain harboring a pGEX-2T expression vector was found to be necessary to prevent recombinant Mb from degrading and to attain its proper folding. The stabilities of recombinant Mbs were generally lower than those of native Mbs, partly due to the absence of post-translational modification. The alpha-Helical content of bullet tuna recombinant Mb at 10 degrees C was the lowest (29.0%) among the recombinant Mbs examined (the values for bluefin tuna and bigeye tuna Mbs being 34.8 and 35.5%, respectively). On the other hand, the stabilities of recombinant Mbs of bluefin tuna and bigeye tuna against denaturants (urea and guanidine hydrochloride) were found to be similar, whereas bullet tuna recombinant Mb exhibited the lowest stability among these Mbs. The pattern of temperature-dependent decrease in the alpha-helical content supported these results.
构建了3种鲭科鱼类重组肌红蛋白（Mb）的表达系统。将这些Mb的稳定性与从慢肌骨骼肌中纯化的天然Mb进行了比较。发现，在培养携带pGEX-2T表达载体的大肠杆菌菌株时添加血红素对于防止重组Mb降解并实现其正确折叠是必要的。重组Mb的稳定性通常低于天然Mb，部分原因是缺乏翻译后修饰。在10℃下，子弹金枪鱼重组Mb的α-螺旋含量在所检测的重组Mb中最低（29.0%）（蓝鳍金枪鱼和大眼金枪鱼Mb的值分别为34.8%和35.5%）。另一方面，发现蓝鳍金枪鱼和大眼金枪鱼重组Mb对变性剂（尿素和盐酸胍）的稳定性相似，而子弹金枪鱼重组Mb在这些Mb中稳定性最低。α-螺旋含量随温度下降的模式支持了这些结果。
