Codon optimization of bacterial luciferase (lux) for expression in mammalian cells
Codon optimization of bacterial luciferase (lux) for expression in mammalian cells
复制标题
DOI：
10.1007/s10295-005-0211-8
复制
发表时间：
2005-03-01
影响因子：
3.4
通讯作者：
Sayler, GS
中科院分区：
文献类型：
--
作者：
Patterson, SS;Dionisi, HM;Sayler, GS
细菌荧光素酶（lux）系统在哺乳动物细胞中的表达将最终产生用于体内监测和诊断技术的新一代生物报告器。过去在哺乳动物细胞中表达细菌荧光素酶的努力仅取得了有限的成果，部分原因是细菌基因的总体表达较低。为了优化表达，我们设计并合成了来自发光杆菌的 luxA 和 luxB 基因的密码子优化版本。为了在体内评估这些基因，创建了稳定的 HEK293 细胞系，其中包含野生型 luxA 和 luxB (WTA/WTB)、密码子优化的 luxA 和野生型 luxB (COA/WTB) 以及 luxA 和 luxB 基因的密码子优化版本 (COA/COB)。尽管这些克隆内的 mRNA 水平保持大致相等，但密码子优化后 LuxA 蛋白水平显着增加。平均而言，密码子优化的 luxA 和野生型 luxB 的生物发光水平增加了六倍以上 [5 x 10(5) vs 2.9 x 10(6) 相对光单位 (RLU)/mg 总蛋白]。两个优化基因的表达（2.7 x 10(7) RLU/mg 总蛋白）进一步增强了生物发光。这些结果显示了在哺乳动物细胞中开发自主发光勒克斯报告系统的潜力。
Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5 x 10(5) vs 2.9 x 10(6) relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7 x 10(7) RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells.