Preparation of artificial 2-, 3-, 4- and 8-domain myoglobins and comparison of their autoxidation rates
Preparation of artificial 2-, 3-, 4- and 8-domain myoglobins and comparison of their autoxidation rates
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DOI：
10.1016/j.febslet.2004.08.028
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发表时间：
2004-09-10
期刊：
影响因子：
3.5
通讯作者：
Suzuki, T
中科院分区：
文献类型：
--
作者：
Kawano, K;Uda, K;Suzuki, T
虽然大多数血红蛋白和肌红蛋白由15 kda的单结构域亚基组成，但结构不寻常的血红蛋白，如Artemia 9结构域和Barbatia 2结构域血红蛋白，在几种无脊椎动物中自然存在。这些血红蛋白似乎是基因复制和融合的结果。利用对黑桃肌红蛋白开放阅读框的cDNA编码，制备了与连续二聚体、三聚体、四聚体和八聚体肌红蛋白（2-、3-、4-和8-结构域）相对应的人工cDNA插入，并将其克隆到pMAL或pQE质粒中。这些人工肌红蛋白和野生型单域肌红蛋白在大肠杆菌中以血红素附着的氧合形式成功表达。用硫酸铵分馏和凝胶过滤纯化部分肌红蛋白，测定自氧化率。带有MBP或六聚His标签的重组野生型肌红蛋白的自氧化率与天然肌红蛋白相当，这表明重组蛋白似乎是正确折叠的，并且n端MBP或His标签对其自氧化率没有影响。另一方面，2域肌红蛋白和3域肌红蛋白的发生率显著降低（分别为单域肌红蛋白的50%和30%）。4-和8-结构域肌红蛋白的发生率与3-结构域肌红蛋白相似。这些结果表明，从结合氧的储存角度来看，人工合成的肌红蛋白的多结构域结构比通常的单结构域肌红蛋白更稳定。(C) 2004年欧洲生化学会联合会。Elsevier B.V.版权所有。
Although most hemoglobins and myoglobins consist of 15-kDa single-domain subunits, structurally unusual hemoglobins, such as Artemia 9-domain and Barbatia 2-domain hemoglobins, occur naturally in several invertebrates. These hemoglobins appear to be the result of gene duplication and fusion. Using cDNA coding for the open reading frame of Aplysia kurodai myoglobin, artificial cDNA inserts corresponding to contiguous dimer, trimer, tetramer and octamer myoglobins (2-, 3-, 4- and 8-domain myoglobins) were prepared and cloned into pMAL or pQE plasmids. These artificial myoglobins and wild-type single-domain myoglobins were successfully expressed in Escherichia coli in the heme-attached, oxygenated form. Myoglobin was purified partially by ammonium sulfate fractionation and gel filtration, and autoxidation rates were examined. The autoxidation rates of recombinant wild-type myoglobins with MBP or hexameric His tag were comparable to those of native myoglobin, suggesting that the recombinant proteins appear to be properly folded and that the N-terminal MBP or His tag does not have an affect on the rate. On the other hand, the rates were significantly decreased in the 2- and 3-domain myoglobins (50% and 30% of the single-domain myoglobins, respectively). The rates for 4- and 8-domain myoglobins were similar to those for 3-domain myoglobin. These results indicate that the artificial poly-domain structure of myoglobin is more stable than the usual single-domain myoglobin from the viewpoint of storage of bound dioxygen. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.