Lone Imtiaz Nisar, Sekeroglu Esin Ozkuru, Kafaz Gkamze Impraimoglou, Al Haija Abed Alkarem Hani Abu, Turker Esra, Batur Tugce, Diril M Kasim
Izmir Biomedicine and Genome Center, 35340, Izmir, Turkey.
Izmir International Biomedicine and Genome Institute, Dokuz Eylül University, Izmir, Turkey.
Mol Biotechnol. 2025 Jul 8. doi: 10.1007/s12033-025-01478-6.
Leukemia inhibitory factor (LIF), a glycoprotein in the interleukin-6 family, is essential for maintaining the pluripotency and self-renewal of mouse embryonic stem cells (mESCs) by activating the JAK/STAT pathway and preventing spontaneous differentiation. Despite its critical role in maintaining the undifferentiated state of ES cells, the high cost of commercially available LIF poses a considerable financial challenge for research laboratories. This financial strain can limit the accessibility and feasibility of high-quality stem cell research, thereby impacting the overall progress in this field. Here, we present an efficient and cost-effective method for recombinant expression and purification of biologically active, non-glycosylated mouse LIF in Escherichia coli, which retains full functionality in mESC culture. Utilizing an N-terminal glutathione S-transferase (GST) tag enhances the solubility of LIF, facilitating rapid purification via glutathione-agarose affinity chromatography. Following purification, LIF is cleaved from the GST tag using HRV 3C protease, resulting in a product ready for direct application in mESC culture. Homemade LIF (HM-LIF) prepared with this protocol effectively maintains mESCs in an undifferentiated state, showing comparable efficacy to commercial LIF. Furthermore, mESCs cultured with HM-LIF retain pluripotency and can differentiate into glutamatergic neurons. This streamlined approach significantly reduces the cost of mESC culture while providing a reliable, high-quality alternative to commercial LIF.
白血病抑制因子（LIF）是白细胞介素-6家族中的一种糖蛋白，通过激活JAK/STAT信号通路并防止自发分化，对于维持小鼠胚胎干细胞（mESC）的多能性和自我更新至关重要。尽管LIF在维持ES细胞未分化状态中起着关键作用，但市售LIF的高成本给研究实验室带来了相当大的经济挑战。这种经济压力会限制高质量干细胞研究的可及性和可行性，从而影响该领域的整体进展。在此，我们提出了一种高效且经济的方法，用于在大肠杆菌中重组表达和纯化具有生物活性的非糖基化小鼠LIF，该LIF在mESC培养中保留了全部功能。利用N端谷胱甘肽S-转移酶（GST）标签可提高LIF的溶解度，便于通过谷胱甘肽-琼脂糖亲和层析进行快速纯化。纯化后，使用HRV 3C蛋白酶从GST标签上切割下LIF，得到的产物可直接用于mESC培养。用该方案制备的自制LIF（HM-LIF）能有效地将mESC维持在未分化状态，其效果与市售LIF相当。此外，用HM-LIF培养的mESC保留了多能性，并且可以分化为谷氨酸能神经元。这种简化的方法显著降低了mESC培养的成本，同时为市售LIF提供了一种可靠、高质量的替代品。
