Dept. of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan.
Biotechnol Prog. 2010 Mar-Apr;26(2):589-94. doi: 10.1002/btpr.351.
Luciferase (Rluc) from the soft coral Renilla reniformis has been widely used as a bioluminescent reporter, and its secreted production has been solely performed in mammalian cells thus far. To make the production more efficient, a series of approaches was attempted to overproduce Rluc extracellularly in Bacillus subtilis. First, Cys124 in the Rluc gene was substituted with Ala. The mutant gene was subsequently incorporated into a pUB110/R6K-based plasmid, consequently, fusing with the P43 promoter and the sacB signal peptide. With the nitrogen-rich medium, B. subtilis strain bearing the plasmid became able to secret a detectable amount of Rluc. Moreover, the secretion signal for the Rluc gene was replaced by the aprN leader peptide with or without the propeptide. The result led to a more than twofold increase in the secreted Rluc. Finally, by enhancing the transcription of the Rluc gene implementing the P43 and spac tandem promoter, it resulted in the secreted Rluc with a yield of 100 mg/L. Overall, this study illustrates a potential strategy for improving the secretion efficiency of heterologous proteins in B. subtilis.
荧光素酶（Rluc）来自软珊瑚 Renilla reniformis，已被广泛用作生物发光报告基因，迄今为止，其分泌产物仅在哺乳动物细胞中表达。为了提高生产效率，人们尝试了一系列方法，试图在枯草芽孢杆菌中体外过量生产 Rluc。首先，将 Rluc 基因中的 Cys124 突变为丙氨酸。随后，突变基因被整合到基于 pUB110/R6K 的质粒中，与 P43 启动子和 sacB 信号肽融合。在富氮培养基中，携带质粒的枯草芽孢杆菌菌株能够分泌可检测量的 Rluc。此外，Rluc 基因的分泌信号被 aprN 前导肽取代，无论是否存在前肽。结果导致分泌的 Rluc 增加了两倍多。最后，通过增强 P43 和 spac 串联启动子的 Rluc 基因转录，使分泌的 Rluc 产量达到 100mg/L。总的来说，这项研究说明了一种在枯草芽孢杆菌中提高异源蛋白分泌效率的潜在策略。
