Proteomics and Cell Biology Lab, Animal Biotechnology Center, National Dairy Research Institute, Karnal, Haryana, India.
PLoS One. 2018 Jun 13;13(6):e0198523. doi: 10.1371/journal.pone.0198523. eCollection 2018.
Leukemia Inhibitory Factor (LIF) is a polyfunctional cytokine, involved in numerous regulatory effects in vivo and in vitro, varying from cell proliferation to differentiation, and has therapeutic potential for treating various diseases. In the current study, a COS-1 cell line overexpressing recombinant Buffalo LIF (rBuLIF) was established. The rBuLIF was purified to homogeneity from the total cell lysate of COS-1 cells using a two-step affinity chromatography. The purified LIF was confirmed by western blot and mass spectrometer (MS/MS). Particularly, high-resolution MS has identified the rBuLIF with 73% of sequence coverage with highest confidence parameters and with the search engine score of 4580. We determined the molecular weight of rBuLIF protein to be 58.99 kDa and 48.9 kDa with and without glycosylation, respectively. Moreover, the purified rBuLIF was verified to be functionally active by measuring the growth inhibition of M1 myeloid leukemia cells, revealing a maximum inhibition at 72 hours and half-maximal effective concentration (EC50) of 0.0555 ng/ml, corresponding to a specific activity of >1.6×10(7) units/mg. Next, we evaluated the effect of rBuLIF on buffalo mammary epithelial cell lines for its role in involution and also identified the IC50 value for BuMEC migrating cells to be 77.8 ng/ml. Additionally, the treatment of MECs (BuMEC and EpH4) displayed significant (P < 0.05) reduction in growth progression, as confirmed by qRT-PCR analysis, suggesting its strong involvement in the involution of the mammary gland in vivo. Thus, we conclude that the glycosylated rBuLIF, purified from COS-1 cells was found to be functionally active as its natural counterpart.
白血病抑制因子（LIF）是一种多功能细胞因子，参与体内和体外的许多调节作用，从细胞增殖到分化不等，具有治疗各种疾病的潜力。在本研究中，建立了一个过表达重组水牛 LIF（rBuLIF）的 COS-1 细胞系。rBuLIF 从 COS-1 细胞的总细胞裂解物中通过两步亲和层析纯化至均一性。通过 Western blot 和质谱仪（MS/MS）确认 LIF。特别是，高分辨率 MS 以最高置信参数和搜索引擎得分 4580 鉴定了 rBuLIF，具有 73%的序列覆盖率。我们确定 rBuLIF 蛋白的分子量分别为 58.99 kDa 和 48.9 kDa，分别为糖基化和非糖基化。此外，通过测量 M1 髓样白血病细胞的生长抑制来验证纯化的 rBuLIF 具有功能活性，在 72 小时时显示最大抑制，半最大有效浓度（EC50）为 0.0555ng/ml，对应于>1.6×10（7）单位/mg 的比活性。接下来，我们评估了 rBuLIF 对水牛乳腺上皮细胞系的作用，以研究其在退化中的作用，并确定了 BuMEC 迁移细胞的 IC50 值为 77.8ng/ml。此外，MECs（BuMEC 和 EpH4）的处理显示出生长进展的显著（P<0.05）减少，这通过 qRT-PCR 分析得到证实，表明其在体内乳腺退化中具有很强的作用。因此，我们得出结论，从 COS-1 细胞中纯化的糖基化 rBuLIF 被发现具有与其天然对应物相同的功能活性。
