A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli
A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli
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DOI：
10.1016/j.jbiotec.2010.12.020
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发表时间：
2011-02-20
影响因子：
4.1
通讯作者：
Ketudat-Cairns, Mariena
中科院分区：
文献类型：
--
作者：
Imsoonthornruksa, Sumeth;Noisa, Parinya;Ketudat-Cairns, Mariena
小鼠胚胎干细胞（mESC）依赖于一种名为白血病抑制因子（LIF）的细胞因子来维持其未分化状态和多能性。然而，由于商业LIF的成本，mESC研究的进展受到限制，并仅限于资金雄厚的实验室。本文介绍了具有生物活性的国产hLIF。将hLIF cDNA克隆到两种不同的载体中，以便在Origami（DE 3）大肠杆菌中产生N-末端His（6）-标签和Trx-His（6）-标签的hLIF融合蛋白。His（6）-hLIF融合蛋白的可溶性不如Trx-His（6）-hLIF融合蛋白。一步固定化金属亲和层析（IMAC）回收高纯度（纯度> 95%）His（6）-hLIF和Trx-His（6）-hLIF融合蛋白，产量分别为100和200 mg/l细胞培养物。Western blot鉴定hLIF融合蛋白，LC/MS/MS鉴定hLIF融合蛋白。hLIF融合蛋白以剂量依赖性方式特异性地促进TF-1细胞的增殖。它们还证明了保留未分化mESC形态的效力，因为它们对mESC标志物（Oct-4、Sox-2、Nanog、SSEA-1和碱性磷酸酶活性）呈阳性。这些结果表明His（6）-hLIF和Trx-His（6）-hLIF融合蛋白的N-末端融合标签不干扰其生物学活性。该表达和纯化方法生产重组hLIF是一种简单、可靠、成本有效和用户友好的方法。(C)2011 Elsevier B. V.保留所有权利。
Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His(6)-tag and Trx-His(6)-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His(6)-hLIF fusion protein was not as soluble as the Trx-His(6)-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (> 95% pure) His(6)-hLIF and Trx-His(6)-hLIF fusion proteins with the yields of 100 and 200 mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His(6)-hLIF and Trx-His(6)-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method. (C) 2011 Elsevier B.V. All rights reserved.