Rathnayaka Tharangani, Tawa Minako, Sohya Shihori, Yohda Masafumi, Kuroda Yutaka
Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
Biochim Biophys Acta. 2010 Sep;1804(9):1902-7. doi: 10.1016/j.bbapap.2010.04.014. Epub 2010 May 7.
Recently, the smallest bioluminescent protein (MW: 19.9 kDa), Gaussia luciferase (GLuc), has been isolated from the marine copepod Gaussia princeps and has attracted much attention as a reporter protein. However, preparation of large quantities of homogeneous natively folded recombinant GLuc appears to be difficult due to its ten cysteines. Here, we report the biophysical characterization of recombinant GLuc expressed using a novel Escherichia coli expression system based on a cold induced expression vector (pCold). Using this system, a large fraction of the protein was expressed in the soluble fraction. GLuc, purified exclusively from the supernatant using nickel affinity chromatography, yielded a large amount of pure GLuc with a native disulfide bond pattern (Soluble-GLuc). Soluble-GLuc had a strong bioluminescence activity and it retained 65% of its activity after 30 min incubation at 95 degrees C. Soluble-GLuc remained fully folded until 40 degrees C, as assessed by circular dichroism; and the thermal denaturation curve was S-shaped, indicating a cooperative transition, with a midpoint temperature of 56 degrees C. These results indicate that both the structure and bioluminescence activity of GLuc remain stable at high temperatures, and they strongly suggest GLuc's potential as a reporter protein.
最近，已从海洋桡足类动物高斯伪镖水蚤中分离出最小的生物发光蛋白（分子量：19.9 kDa）——高斯荧光素酶（GLuc），它作为一种报告蛋白备受关注。然而，由于其含有10个半胱氨酸，制备大量均一的天然折叠重组GLuc似乎很困难。在此，我们报告了使用基于冷诱导表达载体（pCold）的新型大肠杆菌表达系统表达的重组GLuc的生物物理特性。使用该系统，大部分蛋白质在可溶部分表达。仅通过镍亲和色谱从上清液中纯化得到的GLuc，产生了大量具有天然二硫键模式的纯GLuc（可溶性GLuc）。可溶性GLuc具有很强的生物发光活性，在95℃孵育30分钟后仍保留其65%的活性。通过圆二色性评估，可溶性GLuc在40℃之前一直保持完全折叠状态；热变性曲线呈S形，表明是协同转变，中点温度为56℃。这些结果表明，GLuc的结构和生物发光活性在高温下都保持稳定，并且强烈表明GLuc作为报告蛋白的潜力。
