Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli
Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli
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DOI：
10.1016/j.jbiotec.2010.11.010
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发表时间：
2011-01-10
影响因子：
4.1
通讯作者：
Lee, Pyung Cheon
中科院分区：
文献类型：
--
作者：
Kim, Nag-Jong;Choi, Jong Hyun;Lee, Pyung Cheon
构建溶解氧（DO）控制的nor启动子表达载体，通过在大肠杆菌细胞中表达人生长激素（hGH）、增强型绿色荧光蛋白（EGFP）和β -酪氨酸酶，比较其与T7启动子pET22表达载体的表达效率。在大肠杆菌W3110narL（-）中，用T7启动子的5'-未翻译区和核糖体结合位点设计的nor启动子表达载体pRBS以高达32%的细胞总蛋白（TCP）的速率表达了hGH。在hGH中加入β -半乳糖苷酶n端肽标签后，hGH的表达水平进一步提高，达到TCP的42%，与pET-lac:hGH/BL21中TCP的43%的表达水平相当（DE3）。进一步的工程表达载体pRBS（fnr）共表达富马酸/硝酸盐还原酶（fnr），在BL21（DE3）中比pET22表达更多的EGFP。此外，重组β -酪氨酸酶在W3110narL-中成功表达，其表达率接近pRBS（fnr）中TCP的45%。从这些结果来看，本研究开发的do控制的nor启动子系统可以被认为是一种可靠且具有成本效益的蛋白质生产表达系统，特别是在大规模发酵中，作为pET/BL（DE3）系统的替代品。(C) 2010 Elsevier B.V.版权所有
Dissolved oxygen (DO)-controlled nor promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and beta-tyrosinase in Escherichia coli cells. The nor promoter expression vector pRBS, which was engineered with a 5'-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL(-). The expression level of hGH was further enhanced, up to similar to 42% of the TCP, by adding the N-terminal peptide tag of beta-galactosidase to hGH, which was comparable to the expression of similar to 43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant beta-tyrosinase was successfully expressed at a rate of up to similar to 45% of the TCP in pRBS(fnr) in W3110narL-. From these results, the DO-controlled nor promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system. (C) 2010 Elsevier B.V. All rights reserved.