Cell Dynamics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka 563-8577, Japan.
Protein Sci. 2010 Jan;19(1):26-33. doi: 10.1002/pro.279.
A luciferase from the railroad worm (Phrixothrix hirtus) is the only red-emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site-directed mutagenesis, we produced red-emitting mutants with higher activity and better stability. Compared with the wild-type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8-fold in I212L/N351K, 8.4-fold in I212L, and 7.8-fold in I212L/S463R; and the cell-based activities were 3.6-fold in I212L/N351K and 3.4-fold in N351K. The remaining activities after incubation at 37 degrees C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell-based BLI was performed, and the luminescence signal was 3.6-fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.
一种来自铁路蠕虫（Phrixothrix hirtus）的荧光素酶是自然界中唯一的红色发射生物发光酶，它在多色荧光素酶检测和生物发光成像（BLI）中具有优势。然而，由于其活性和稳定性低，它并没有在科学或工业应用中得到广泛应用。通过使用定点突变技术，我们产生了具有更高活性和更好稳定性的红色发射突变体。与野生型（WT）相比，表达突变型荧光素酶的培养哺乳动物细胞提取物的发光活性在 I212L/N351K 中为 9.8 倍，在 I212L 中为 8.4 倍，在 I212L/S463R 中为 7.8 倍；细胞基础活性在 I212L/N351K 中为 3.6 倍，在 N351K 中为 3.4 倍。在 37°C 孵育 10 分钟后，I212L/S463R 的剩余活性为 50.0%，I212L 的剩余活性为 31.8%，I212L/N351K 的剩余活性为 23.0%，而 WT 的剩余活性仅为 5.2%。为了证明 I212L/N351K 的应用，进行了基于细胞的 BLI，其发光信号比 WT 高 3.6 倍。这些结果表明，这些突变体可能会提高该信号在生物测定和 BLI 中的实用性。
