Liu Huihui, Wang Xiaolin, Shi Shuang, Chen Yingyu, Han Wenling
Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Medical Immunology, Ministry of Health, Beijing, China; Peking University Center for Human Disease Genomics, Peking University Health Science Center, Beijing, China.
Peking University Center for Human Disease Genomics, Peking University Health Science Center, Beijing, China.
Protein Expr Purif. 2015 Sep;113:1-7. doi: 10.1016/j.pep.2015.05.004. Epub 2015 May 12.
FAM19A4 is a novel potential cytokine identified by our group, which can chemoattract macrophages, promote phagocytosis against zymosan and increase reactive oxygen species (ROS) release. To further explore the role of FAM19A4 in immune system, abundant recombinant protein with high quality is indispensable. For efficient production of FAM19A4, we used an improved CHO-S cell expression system on the basis of pMH3 vector containing GC-rich regions which were novel ubiquitous chromatin opening elements (UCOEs). We selected CHO-S cells stably expressing FAM19A4 with G418 and screened cell clones with high level of FAM19A4 expression by immune blot and his-ELISA, adapted cell clones to serum-free suspension culture. Afterwards, we obtained the highest FAM19A4 expressing cell clone (2#) through 40 ml batch culture. We optimized the fed-batch culture condition and discovered the final cell viability was critical for FAM19A4 production successfully. Then we scaled 2# clone up to 3 L in fed-batch culture and obtained 22 mg (7.33 mg/L, averagely) endotoxin free FAM19A4 protein with purity over 95% using Ni affinity chromatography and size exclusion chromatography. The final yield was increased 3.6-folds compared to that of our previously reported transient system. Besides, the purified FAM19A4 protein showed chemotactic activity on macrophages. In summary, we developed a stable optimized fed-batch CHO-S cell system to produce FAM19A4, which not only provided sufficient bioactive FAM19A4 protein for further research but also offered an efficient strategy for other recombinant protein production.
FAM19A4是我们团队鉴定出的一种新型潜在细胞因子，它可以趋化巨噬细胞，促进对酵母聚糖的吞噬作用，并增加活性氧（ROS）的释放。为了进一步探索FAM19A4在免疫系统中的作用，高质量的大量重组蛋白是必不可少的。为了高效生产FAM19A4，我们在含有富含GC区域（即新型普遍存在的染色质开放元件（UCOE））的pMH3载体的基础上，使用了改进的CHO-S细胞表达系统。我们用G418筛选出稳定表达FAM19A4的CHO-S细胞，并通过免疫印迹和组氨酸ELISA筛选出FAM19A4表达水平高的细胞克隆，使细胞克隆适应无血清悬浮培养。之后，我们通过40 ml分批培养获得了表达FAM19A4最高的细胞克隆（2#）。我们优化了补料分批培养条件，发现最终细胞活力对成功生产FAM19A4至关重要。然后我们将2#克隆扩大到3 L补料分批培养，使用镍亲和层析和尺寸排阻层析获得了22 mg（平均7.33 mg/L）内毒素含量低于检测限且纯度超过95%的FAM19A4蛋白。最终产量比我们之前报道的瞬时表达系统提高了3.6倍。此外，纯化的FAM19A4蛋白对巨噬细胞显示出趋化活性。总之，我们开发了一种稳定优化的补料分批CHO-S细胞系统来生产FAM19A4，这不仅为进一步研究提供了足够的具有生物活性的FAM19A4蛋白，也为其他重组蛋白的生产提供了一种有效的策略。
