Expression of human il-15 in Pichia pastoris final V

分类： 生物学 >> 生态学 提交时间： 2017-11-17

Wei Sun Yunxin Lai Hongbo Li Tao Nie Ye Kuang Xiaofeng Tang Kuai Li P. Rod Dunbar Aimin Xua Peng Li Donghai Wu

摘要： Interleukin-15 (IL-15) is a pleiotropic cytokine and a member of the four α-helix bundle family of cytokines which include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-15 exhibits a broad biological activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. In this study, a DNA fragment containing the mature human IL-15 sequence was cloned into pPICZaA vector, generating a fusion protein with the alpha factor signal sequence in the Nterminus 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 recombinant human IL-15 (rhIL-15) production were identified, which secrete as much as 75 mg/L rhIL-15 after 3 days of induction by methanol. The rhIL-15 was purified by Ni+-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-15. Mass spectrometry and MALDI-TOF-TOF analysis showed the purified rhIL-15 had larger molecular weights than expected, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-15 proteins was measured by its ability to enhance cellular proliferation of CTLL-2 and NK cells. The results demonstrate that the experimental procedure we have reported here can produce a large amount of active recombinant human IL-15 from Pichia pastoris.

同行评议状态:待评议

 点击量 2682  下载量 1504  评论