Jamwal Shradha, Ansari Shama, Malakar Dhruba, Kaushik Jai Kumar, Kumar Sudarshan, Mohanty Ashok Kumar
Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, India.
Indian Council of Agricultural Research-Indian Veterinary Research Institute, Mukteshwar, India.
J Genet Eng Biotechnol. 2022 Mar 16;20(1):47. doi: 10.1186/s43141-022-00328-1.
Leukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli).
The BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity.
The present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.
白血病抑制因子（LIF）是一种多功能细胞因子，在植入、骨重塑和造血等不同生物学过程中发挥多种作用。由于对培养条件缺乏适当了解，水牛胚胎干细胞（buESCs）难以培养。LIF是维持胚胎干细胞多能性的重要因素之一，在水牛胚胎干细胞的建立中使用了来自小鼠和人类来源的商业LIF。来自外源的LIF不能长期维持buESCs的多能性和增殖，这是由LIF上结合位点的差异造成的；因此，添加水牛特异性LIF的培养基可能通过改善培养条件来提高buESCs的效率。LIF的高成本是限制buESCs研究的另一个主要缺点，从而限制了水牛干细胞的应用范围。已经开发了各种方法在原核系统中生产人和小鼠LIF。然而，水牛白血病抑制因子（BuLIF）尚未在原核系统中生产。在此，我们描述了一种在大肠杆菌（E. coli）中表达和纯化具有生物活性的BuLIF的简单策略。
将来自水牛（Bubalus bubalis）的BuLIF cDNA克隆到pET22b(+)中，并在大肠杆菌Lemo-21(DE3)中表达。BuLIF的表达定向到大肠杆菌的周质空间，导致可溶性重组蛋白的形成。通过一步固定金属亲和色谱（IMAC色谱）进行BuLIF的纯化，纯度≥95%。通过蛋白质免疫印迹法确认重组蛋白，并通过质谱法进行鉴定。通过MTT增殖试验在小鼠髓性白血病细胞（M1细胞）上测定重组BuLIF的生物学活性。添加BuLIF以剂量依赖性方式增加了受刺激的M1细胞对MTT的还原。BuLIF诱导M1细胞形成巨噬细胞样结构，这些细胞吞噬荧光乳胶珠。重组BuLIF成功维持了水牛胚胎干细胞（buESCs）的多能性，并且对干细胞标志物如Oct-4、Sox-2、Nanog和碱性磷酸酶活性呈阳性。
本研究展示了一种通过单步纯化在大肠杆菌中生产生物活性BuLIF的简单方法。BuLIF有效地维持了水牛胚胎干细胞的多能性。因此，这种纯化的BuLIF可用于干细胞研究、生物医学和农业研究。
