High-Level Production of Human Leptin by Fed-Batch Cultivation of Recombinant Escherichia coli and Its Purification
High-Level Production of Human Leptin by Fed-Batch Cultivation of Recombinant Escherichia coli and Its Purification
复制标题
重组大肠杆菌流加培养高水平生产人瘦素及其纯化
DOI：
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发表时间：
1999
影响因子：
4.4
通讯作者：
S. Lee
中科院分区：
文献类型：
--
作者：
K. Jeong;S. Lee
摘要人类瘦素是一种由脂肪细胞分泌的16 kDa(146个氨基酸)的蛋白质，影响体重的动态平衡。为了获得瘦素的高水平表达，我们在大肠杆菌BL21(DE3)中，在T7强诱导启动子的作用下，表达了人瘦素基因。重组瘦素在大肠杆菌中以包涵体形式表达，其含量高达总蛋白的54%。为了大量生产重组人瘦素，进行了pH恒定流加培养。在600 nm、30、90和140 nm三种不同的细胞光密度下诱导Leptin的表达。当OD600为90时，细胞产生瘦素的量为9.7g/L(占总蛋白的37%)。经过包涵体分离、变性复性、阴离子交换层析等简单纯化步骤，从50ml培养物中可获得纯度大于90%的瘦素144.9 mg，回收率为41.1%。
ABSTRACT Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human obesegene coding for leptin was expressed in Escherichia coliBL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in E. coli, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD600), 30, 90, and 140. When cells were induced at an OD600 of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%.
影响因子：
56.9
作者：
HALAAS, JL;GAJIWALA, KS;FRIEDMAN, JM
通讯作者：
FRIEDMAN, JM
重组瘦素的生产和重折叠。
影响因子：
2.7
作者：
Zamorano,PL;DeSevilla,L;Mahesh,VB;Brann,DW
通讯作者：
Brann,DW