Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging
Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging
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DOI：
10.1002/pro.279
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发表时间：
2010-01-01
期刊：
影响因子：
8
通讯作者：
Ohmiya, Yoshihiro
中科院分区：
文献类型：
--
作者：
Li, Xueyan;Nakajima, Yoshihiro;Ohmiya, Yoshihiro
来自铁路蠕虫（Phrixothrix hirtus）的荧光素酶是自然界中唯一的发红光的生物发光酶，其在荧光素酶测定和生物发光成像（BLI）中是有利的。然而，由于其低活性和稳定性，它在科学或工业应用中没有广泛使用。通过定点突变，我们获得了具有更高活性和更好稳定性的发红光突变体。与野生型（WT）相比，来自表达突变荧光素酶的培养哺乳动物细胞提取物的发光活性在I212 L/N351 K中为9.8倍，在I212 L中为8.4倍，在I212 L/S463 R中为7.8倍;并且基于细胞的活性在I212 L/N351 K中为3.6倍，在N351 K中为3.4倍。在37 ℃孵育10分钟后，I212 L/S463 R的剩余活性为50.0%，I212 L为31.8%，I212 L/N351 K为23.0%，但WT仅为5.2%。为了证明I212 L/N351 K的应用，进行了基于细胞的BLI，并且发光信号比WT高3.6倍。这些结果表明，突变体可能会提高这种信号在生物测定和BLI的实用性。
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.