Chang T-S, Wu W-J, Wan H-M, Shiu T-R, Wu W-T
Department of Chemical Engineering, National Tsing Hua University, 30043, Hsinchu, Taiwan.
Appl Microbiol Biotechnol. 2003 May;61(3):234-9. doi: 10.1007/s00253-003-1252-4. Epub 2003 Feb 26.
The GlnAP2 element has been proved to be an effective and inducible-by exogenous acetate-promoter in Escherichia coli with glnL/pta double mutations. Based on this feature, a single-copy expression vector was constructed via coupling of the glnAP2 promoter-regulated T7 RNA polymerase gene and the T7-promoter-controlled lacZ gene on a bacterial artificial chromosome. After induction with 20 mM potassium acetate, the glnL/pta double mutant E. coli harboring the single-copy plasmid produced 47,500 Miller units of beta-galactosidase activity. This high level expression, corresponding to 27% of total cell protein, was comparable to that determined with the commercial multi-copy expression vector, pET-14b, in strain E. coli Tuner (DE3) (64,300 Miller units, 41% of total cell protein). Moreover, this single-copy expression vector could be maintained for at least 150 generations even in the presence of inducers. In contrast, the multi-copy expression vector was extensively lost after induction. The results indicate that the single-copy expression system has the potential for high-level heterologous protein production for industrial applications.
已证明GlnAP2元件在具有glnL/pta双突变的大肠杆菌中是一种有效的、可被外源乙酸盐诱导的启动子。基于这一特性，通过将glnAP2启动子调控的T7 RNA聚合酶基因与细菌人工染色体上T7启动子控制的lacZ基因偶联，构建了单拷贝表达载体。用20 mM乙酸钾诱导后，携带单拷贝质粒的glnL/pta双突变大肠杆菌产生了47,500个米勒单位的β-半乳糖苷酶活性。这种高水平表达相当于总细胞蛋白的27%，与在大肠杆菌Tuner (DE3)菌株中使用商业多拷贝表达载体pET-14b所测定的水平相当（64,300个米勒单位，占总细胞蛋白的41%）。此外，即使在存在诱导剂的情况下，这种单拷贝表达载体也能维持至少150代。相比之下，多拷贝表达载体在诱导后大量丢失。结果表明，单拷贝表达系统具有在工业应用中高水平生产异源蛋白的潜力。
