Li Hongbo, Li Na, Gao Xuefei, Kong Xiangping, Li Shiwu, Xu Aimin, Jin Shouguang, Wu Donghai
The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
Protein Expr Purif. 2011 Dec;80(2):185-93. doi: 10.1016/j.pep.2011.08.027. Epub 2011 Aug 31.
Interleukin-3 (IL-3) is a hematopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hematopoietic cells. A DNA fragment containing the mature human IL-3 sequence was cloned into pPICZαA, generating a fusion protein with the alpha factor signal sequence in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level rhIL-3 production were identified, secreting as much as 26mg/L rhIL-3 after 4days of induction by methanol in flask. The rhIL-3 was purified by Ni(+)-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-3 preparation at about 21mg/L. Mass spectrometry and MALDI-TOF-TOF analysis of the purified rIL-3 showed molecular weights of 18995.694Da and 22317.469Da, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-3 proteins was confirmed by its ability to support ba/f3 cells proliferation and activate the ERK signaling pathways. The results demonstrate that the experimental procedure we have developed can produce a large amount of active recombinant human IL-3 from P. pastoris.
白细胞介素-3（IL-3）是一种造血生长因子，参与多能造血细胞的存活、增殖和分化。将包含成熟人IL-3序列的DNA片段克隆到pPICZαA中，产生一种在N端带有α因子信号序列且在C端带有6×His以及c-Myc标签的融合蛋白。所得质粒整合到毕赤酵母菌株X-33的基因组中。鉴定出具有高水平重组人IL-3（rhIL-3）产生的重组酵母转化体，在摇瓶中用甲醇诱导4天后可分泌高达26mg/L的rhIL-3。rhIL-3通过Ni(+)-NTA亲和层析进行纯化，随后进行DEAE阴离子交换，以约21mg/L的产量获得超过95%的高纯度rhIL-3制剂。对纯化的重组IL-3进行质谱分析和基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-TOF）分析，由于N-糖基化程度不同，显示分子量分别为18995.694Da和22317.469Da。rhIL-3蛋白的生物学活性通过其支持ba/f3细胞增殖和激活细胞外信号调节激酶（ERK）信号通路的能力得到证实。结果表明，我们开发的实验方法能够从毕赤酵母中大量生产有活性的重组人IL-3。
