Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner
Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner
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DOI：
10.1016/j.pep.2010.04.002
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发表时间：
2010-09-01
影响因子：
1.6
通讯作者：
Scheper, Thomas
中科院分区：
文献类型：
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作者：
Tomala, Magda;Lavrentieva, Antonina;Scheper, Thomas
白血病抑制因子（LIF）是一种多功能细胞因子，在体内和体外具有多种调节作用。在干细胞培养中，它是维持胚胎和诱导多能干细胞多能性的必要培养基补充剂。关于这些细胞的大规模培养，LIF是高质量和高数量的需要，并且代表了培养基的主要成本决定因素（90%）。在这份报告中，我们描述了一种新的生产和纯化过程中的人白血病抑制因子（hLIF）的重组大肠杆菌文化。将hLIF克隆到pET 32b中，并以与硫氧还蛋白融合的可溶性蛋白形式表达。在基于膜吸附技术的纯化后，融合蛋白用TEV蛋白酶切割。随后通过阳离子交换色谱法纯化释放的可溶性hLIF，并使用小鼠胚胎和诱导多能干细胞的悬浮培养物成功地测试其生物活性。我们生产重组hLIF的新方案非常适合和有效地用于通过与增溶伴侣硫氧还蛋白融合表达来生产难溶性蛋白。(C)2010年爱思唯尔公司All rights reserved.
Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin. (C) 2010 Elsevier Inc. All rights reserved.