Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli
Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli
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DOI：
10.1016/j.pep.2004.04.007
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发表时间：
2004-07-01
影响因子：
1.6
通讯作者：
Sung, MH
中科院分区：
文献类型：
--
作者：
Jeong, KJ;Choi, JH;Sung, MH
研究了使用组成型启动子系统通过补料分批培养重组大肠杆菌来高水平生产人瘦素。为了组成型表达编码人瘦素的肥胖基因，使用从托氏地芽孢杆菌的D-氨基酸转氨酶基因克隆的强组成型HCE启动子。为了开发最佳的宿主载体系统，对几种不同的重组大肠杆菌菌株的瘦素生产进行了比较。在烧瓶培养中，大肠杆菌 FMJ123（一种 rpoS 突变菌株）显示出最高水平的瘦素产量（占总蛋白的 41%）。通过比较几种不同rpoS(-)和rpoS(+)菌株中瘦素的表达水平，可以得出结论：rpoS突变对瘦素的组成型产生有正向影响。为了大规模生产人瘦素，使用三种不同的补料溶液（化学成分确定的补料溶液、含有酵母提取物的补料溶液和含有酪蛋白氨基酸的补料溶液）进行重组大肠杆菌 FMJ123 的补料分批培养。其中，使用含有酪蛋白氨基酸的补料溶液可产生高达 2.1 g/L 的瘦素，分别比使用化学成分确定的补料溶液和含有酵母提取物的补料溶液所获得的瘦素高 21 倍和 1.8 倍。这些结果表明，HCE 启动子可用于以组成型方式有效生产瘦素以及很可能的其他重组蛋白。 (C) 2004 Elsevier Inc. 保留所有权利。
High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E coli strains were compared for leptin production. In flask cultures, E coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS(-) and rpoS(+) strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E coli FMJ123 were carried out using three different feeding solutions-chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 21- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner. (C) 2004 Elsevier Inc. All rights reserved.