Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea.
Research Institute for Convergence of Basic Science, Hanyang University, Seoul 04763, Republic of Korea.
Biosensors (Basel). 2024 Nov 1;14(11):528. doi: 10.3390/bios14110528.
luciferase (Luc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly enhance bioluminescence (BL) intensity. Among the mutated proteins expressed in bacteria, five individual mutations (M60L, K88Q, F89Y, I90L, or S103T) independently increased BL intensity by 1.8 to 7.5-fold compared to wild-type Luc in the presence of coelenterazine substrates. Remarkably, the combination of all five mutations in Luc (designated as Luc5) resulted in an unexpected 29-fold enhancement in BL intensity. Subsequent evaluation of the Luc5-secreted reporter in transfected mammalian cells confirmed its superior BL performance across multiple cell lines. These findings suggest that the mutated residues are likely crucial for enhancing BL intensity in Luc, supporting its potential to serve as a highly sensitive biosensor or reporter for a wide range of biological applications.
荧光素酶（Luc）是一种卓越的分泌型荧光素酶，广泛应用于基于细胞的报告基因检测。通过对不同桡足类荧光素酶序列进行序列引导的突变，并进行比对分析，我们确定了一些关键的氨基酸残基，这些氨基酸残基能够显著提高生物发光（BL）强度。在细菌中表达的突变蛋白中，有五个单独的突变（M60L、K88Q、F89Y、I90L 或 S103T）在存在腔肠素底物的情况下，与野生型 Luc 相比，BL 强度分别独立提高了 1.8 到 7.5 倍。值得注意的是，Luc 中的所有五个突变（命名为 Luc5）的组合导致 BL 强度意外增强了 29 倍。随后在转染的哺乳动物细胞中对 Luc5 分泌型报告基因进行评估，证实了其在多种细胞系中具有优越的 BL 性能。这些发现表明，突变残基可能对增强 Luc 的 BL 强度至关重要，支持其作为一种高灵敏度生物传感器或报告基因，应用于广泛的生物学研究。
