Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system
Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system
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DOI：
10.1016/s0032-9592(99)00013-8
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发表时间：
1999-10-01
影响因子：
4.4
通讯作者：
Wong, WKR
中科院分区：
文献类型：
--
作者：
Sivakesava, S;Xu, ZN;Wong, WKR
携带质粒 pWKW2 或 lacUV5par8EGF（均编码人表皮生长因子 (hEGF)）的重组大肠杆菌 JM101 菌株用于发酵，以优化 hEGF 的分泌水平。针对重组蛋白的体积生产，对培养基组成、诱导剂水平、诱导生长阶段和培养条件进行了优化。选择以 5 g/l 葡萄糖和胰蛋白胨作为氮源的 MMBL 培养基。发现大肠杆菌 JM101[pWKW2] 的异丙基-β-D-硫代半乳糖苷 (IPTG) 浓度为 0.1 mM，大肠杆菌 K-12 JM101[lacUV5par8EGF] 的浓度为 0.2 mM，可提供最佳的 hEGF 生产水平。当培养物在对数中期诱导时，hEGF 的体积产量最大。生长温度对hEGF产量有显着影响。开发了一种简单的连续补料分批培养 E. call JM101[pWKW2] 的工艺。在连续补料分批培养中获得的hEGF最大分泌浓度为325毫克/升，而在分批培养中为175毫克/升。通过 SDS-PAGE 和免疫印迹证实了连续补料分批培养产生的 hEGF。 (C) 1999 Elsevier Science Ltd. 保留所有权利。
Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-beta-D-thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. call JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting. (C) 1999 Elsevier Science Ltd. All rights reserved.